A Floral Ontogenetic Study in Croton (Euphorbiaceae) with Special Emphasis on the Evolution of Petals

A floral ontogenetic study in Croton (Euphorbiaceae) with special emphasis on the evolution of petals.

Stuart Ritchie MSc Biodiversity and Taxonomy of Plants 2017/2018

Thesis submitted in partial fulfilment for the MSc in the Biodiversity and Taxonomy of Plants

Acknowledgments

Sincerest thanks to my project supervisors Pakkapol Thaowetsuwan and Louis Ronse De Craene for their support, guidance and knowledge. I look forward to working together to publish the results presented here. Thank you also to Louis and all staff members at the Royal Botanic Garden Edinburgh who contributed to the MSc Biodiversity and Taxonomy of Plants over the last year. Learning from such respected botanists in a world class botanic garden has been a truly life changing experience. My love and appreciation to Emmy for her constant love and support, which has no limits, and to Mum, Dad and Gran J for their love and encouragement.

Of course, thank you to my friends who have made the last year so special. It has been a great pleasure spending it with such a diverse group of people, all united by a love of plants!

Finally, thank you The Scottish International Education Trust who awarded me a grant towards tuition fees and to The Stirlingshire Educational Trust for their grant towards travel expenses. Without these grants and generous contributions towards my course fees, travel and living expenses from family members, accepting a place on the course would not have been possible. Thank you.

Contents

Abstract 1.Introduction 1.1. The history and principles of comparative morphology including arguments for its central role in the study of floral evolution.

1.2. Introduction to the angiosperm perianth with an emphasis on the evolution of petals.

1.3. Petal-like organs in the basal angiosperms.

1.4. Evolution of the core eudicot perianth with emphasis on petals.

1.5. Genetic control of floral organ identity.

1.6. An introduction to Croton: Diversity and ecology, morphology and taxonomy. 1.6.1. Diversity and Ecology 1.6.2. Morphology 1.6.3. Taxonomy

1.7. Research aims.

2.Methods 2.1. Dissection, Critical Point Drying and Scanning Electron Microscopy 2.2. Tissue clearing and viewing 3. Results 3.1. Organography of C. alabamensis 3.2. Organogeny in male flowers of C. alabamensis 3.3. Organogeny in female flowers of C. alabamensis 3.4. Perianth vasculature of male and female flowers of C. alabamensis 3.5. Organography of C. schiedeanus 3.6. Organogeny in male flowers of C. schiedeanus 3.7. Organogeny in female flowers of C. schiedeanus

3.8. Perianth vasculature of male and female flowers of C. schiedeanus 3.9. Organography of C. chilensis 3.10. Organogeny in male flowers of C. chilensis 3.11. Organogeny in female flowers of C. chilensis 3.12. Perianth vasculature of male and female flowers of C. chilensis 4. Discussion 4.1. Petal evolution in Croton 4.2. The identity of nectaries in Croton 4.3. Evolution of the androecium in Croton

5. Conclusions

6. References

Abstract

Background and Aims: Croton is a megadiverse genus of over 1200 species within the family Euphorbiaceae. The unisexual flowers of Croton species show a particularly high diversity in the perianth, number and position of stamens and in the floral nectaries. In most Croton taxa, male flowers have well-developed petals which are generally reduced or absent in female flowers. However, in two New World Croton sections, petals are equally well developed in both male and female flowers. Due to a lack of developmental studies within the genus, the identity of these petals and of the filamentous petals occurrent throughout the genus remains unresolved. This study seeks to answer three main questions: Are the petals in male Croton flowers of tepaline origin like the sepals, or are they derived from sterilised and transformed stamens? Are the well-developed petals in female flowers of C. alabamensis and C. schiedeanus homologous to those in male flowers? What are the implications for the evolutionary identity of the filamentous petals found in C. chilensis and throughout the genus at large?

Methods: The floral ontogeny and morphology of three Croton species, C. alabamensis, C. schiedeanus and C. chilensis were studied and examined using scanning electron microscopy. Perianth vasculature was examined using clearing and staining techniques.

Key Results: The petals in male and female flowers of C. alabamensis and C. schiedeanus are confirmed as bracteopetals and are not derived from transformed staminodes. The floral ontogeny of C. chilensis supports earlier hypotheses that the filamentous petals in female flowers are reduced petals. These are shown to have a delayed development. The development of the androecium is highly varied in the three species studied. Stamen initiation is centripetal in C. schiedeanus leading to an obdiplostemonous configuration at maturity. The number, size and shape of floral nectaries is variable between species. They develop late in the floral ontogeny indicating that they are of receptacular origin.

Discussion: The case for a process of repeated reduction of female petals throughout the genus is presented and discussed with regard to heterochrony, genetics and spatial pressures imposed during the floral ontogeny. The identity of the floral nectaries of receptacular origin is discussed. The special case of centripetal obdiplostemony in C. schiedeanus is discussed in the context of the angiosperms at large.

1. Introduction

1.1. The history and principles of comparative morphology including arguments for its central role in the study of floral evolution.

Goethe coined the term ‘morphology’. He also introduced the concept of the ‘leaf’ (phyllome of Troll) as the primary plant organ, which undergoes ‘metamorphosis’ to create each of the distinct floral organs that we now call sepals, petals, stamens and carpels (Goethe, 1790; Claßen-Bockhoff, 2001). Later, Takhtajan with an understanding of Darwinian evolution and early genetic studies, discussed the evolutionary origin of floral organs from modified laminar, leaf-like structures through developmental processes acting on the reproductive shoots of a seed-fern-like ancestor (Takhtajan, 1976; Takhtajan, 1991). Since Takhtajan, several floral morphologists have argued that once the ancestral angiosperm flower had evolved, all extant floral diversity could be generated through the modification of a few major developmental processes, as influenced by a combination of external and internal pressures. The first is heterochrony, a term introduced by Haeckel which has since been modified by several morphologists (Haeckel, 1875). It is a change in the timing or rate of development of ancestral features, such that the modified ontogeny becomes fixed in descendants (Li and Johnston, 2000). The second is heterotopy, defined as a change in the position of structures during development. Finally, there is homeosis, which whilst variously defined means transference of attributes of one structure, to the position normally occupied by a structure with different attributes (Ronse De Craene, 2003). When studied within the context of evolutionary history, evolutionary genetics (Evo-Devo) and plant physiology, knowledge of developmental processes provides great potential for understanding the evolution of flowers and floral diversity (Ronse De Craene, 2018). Li and Johnston (2000) wrote that these processes can be summarised into four elements governing the development of flowers. These are size, shape, timing and rate. Ronse De Craene (2018) argued that heterotopy is a superfluous term as it can both be caused by homeosis, and is a direct consequence of heterochrony due to the change in available space on the floral meristem at the time of organ initiation. He argues that pressure is an element overlooked by Li and Johnston in that it governs the shape and size of floral organs. As such, it is suggested the combination of just three factors, time, size and pressure acting during floral development can explain the resulting morphology (Ronse De Craene, 2018).

From a Neo-Darwinian perspective, it is reasonable to believe that that changes in floral developmental processes must be preceded by genetic mutations, which subsequently become fixed due to selective forces imposed by pollinators. However, it is recognised that different floral morphologies may be created based on similar genetics. Most of the genes that are well studied are floral organ identity genes, the expression of which may be shifted in space-time, resulting in homeosis. These genes do not govern the position of organs within flowers (Jaramillo and Kramer, 2007). Instead, of central importance to the study of floral evolution is an understanding of how mechanical forces (pressure), geometry (size and shape) and growth (time) act as cues for cellular behaviour and the initiation of different genetic pathways. Several inter-disciplinary studies have demonstrated the importance of these factors, particularly pressure, in regulating other processes during plant development. For example, mechanical pressures are responsible for auxin transport and accumulation during all stages of plant growth. The phytohormone auxin is the primary regulator of growth patterns in plants. It induces cell growth at very specific sites such as organ primordia during flower and shoot development (Nakayama et al., 2012). Shoot morphogenesis, which depends on coordinated growth of the cellular microtubule cytoskeleton, is also closely regulated by mechanical pressures at all stages (Hamant et al., 2008). Hamant et al. (2008) demonstrated that in the absence of both pressure and mechanically regulated auxin gradients, Arabidopsis thaliana meristems fail to develop any form of tissue organisation. Supported by many observations like these, it is clear that floral development is not solely regulated by genetic systems. Biomechanical forces with a degree of stochasticity, both external and internal, in combination with biochemical regulation, appear to have a central role.

Ronse De Craene (2018) suggested that all extant floral diversity has been generated through cycles of change in developmental processes which are limited by external pressures (pollinators) and spatial constraints within flowers, and subsequent periods of genetic stabilization. Such hypotheses are becoming increasingly supported by evidence from multiple fields of developmental biology. They emphasise the importance of comparative morphology at all stages of floral development to understanding the evolution of flowers.

1.2. Introduction to the angiosperm perianth with an emphasis on the evolution of petals Petals are a typical feature of eudicot angiosperms. Whilst the majority of angiosperms have flowers with a perianth, which encloses and protects the reproductive organs, the core eudicot perianth has differentiated into two whorls with different anatomy and function. Historically, petals have been loosely defined as the visually attractive organs making up the corolla, or inner whorl of a differentiated perianth. The ordinarily green organs in the outer whorl are termed sepals and collectively form a calyx. The calyx functions in protection of the inner organs during development (Endress, 2011).

It is evident that non-homologous floral organs have been described as petals throughout the history of botanical research. Reviewers have attempted to find a common definition for the term by examining the meaning in the context of developmental genetics, morphology, floral ontogeny, and through historical usage. However, the conclusion is that petals cannot be homologised across the breadth of angiosperm diversity (Ronse De Craene, 2007; Ronse De Craene and Brockington, 2013). Whilst this means we are unable to determine if the petals of one taxon are homologous to those of another simply by observing mature flowers, the nature of petals can be determined through research into developmental variation between plants at different taxonomic levels.

Whilst the details concerning the origins of the angiosperm flower are beyond the scope of this study, it is generally thought that the perianth of the ancestral angiosperm evolved from the inclusion of leaf-like organs, or phyllomes, into the earliest flowers (Endress, 2008; Endress and Doyle, 2009; Sauquet et al., 2017). Although these deductions were made using a very different scientific methodology, they are inspired by the musings of Goethe some 230 years ago. If the ancestor of all extant angiosperms had a perianth, then it is reasonable to conclude that the tepals of the basal angiosperms and monocots are homologous with that found in such an ancestor (Bateman et al., 2006; Doyle, 2008; Specht and Bartlett, 2009). However, in the eudicots, two ‘classes’ of petals have been recognised to describe organs with contrasting evolutionary origins, ‘bracteopetals’ and ‘andropetals’ (Ronse De Craene and Brockington, 2013). Bracteopetals evidently are derived from the ancestral bract-like tepals. In contrast, andropetals are derived from sterilized, modified stamens. Andropetals evolved as a means of reinventing attractive structures in plant lineages which lost their ‘true-petals’ at some stage during their evolution. Until recently, it was generally accepted that the petals of most core eudicots are andropetals (Ronse De Craene and Smets, 2001). This belief was largely derived

10 from the study of groups in the basal grade at the base of the eudicot phylogeny, particularly the Ranunculales (Angiosperm Phylogeny Group, 2016). The nectar leaves which occur in several families in this order and are particularly prominent in the Ranunculaceae, have been used as direct evidence for a staminodial origin of petals in the eudicots (Endress, 1995). This implies a loss of true bracteopetals in the ancestor of all extant eudicots. However, this hypothesis has been the subject of considerable scrutiny over the last two decades and is no longer universally accepted.

To understand the occurrence and evolution of these two types of petals, it is first necessary to discuss the evolutionary origin of the eudicot differentiated perianth in the context of the angiosperm phylogeny.

1.3. Petal-like organs in the basal angiosperms In the basal angiosperms which encompass the ANA-grade (Amborellales, Nymphaeales and Austrobaileyales), Chloranthales and Magnoliids (Canellales, Magnoliales, Piperales and Laurales) (Angiosperm Phylogeny Group, 2016), most taxa share flowers with a spiral or whorled phyllotaxis in which there is a transitional morphological gradient in the floral organs from the exterior to interior of flowers. This is particularly evident in taxa such as Illicium henryi Diels (Schisandraceae) and Austrobaileya scandens White (Austrobaileyaceae), which have floral organs arranged in a spiral which transition from bracts to petal-like organs (Fig. 1A,B). In this case, all the perianth parts have a duel function in protection and attraction; the terms petal and sepal have no relevance. These duel functioning organs which form the perianth in most basal angiosperms and petaloid monocots are best referred to as tepals. Whilst almost ubiquitously known as a core eudicot character, a differentiated perianth occurs in several basal angiosperm and monocot families. Within the Magnoliaceae and Annonaceae, flowers with a differentiated perianth arranged in three trimerous whorls are common. The outer whorl consists of three sepaloid sepals and the two inner whorls each contain three petaloid petals (Ronse De Craene, 2010). In Magnolia grandiflora L. (Magnoliaceae) perianth differentiation is weak. However, there is a clear contrast in morphology between the organs in the outermost perianth whorl and those in the innermost whorl, here referred to as sepals and petals respectively (Fig. 1C). The case for perianth differentiation in the Magnoliaceae is supported by ontogenetic evidence which suggests that the outermost whorl is true perianth and not a reinvention of staminodial petals (Xu, 2006; Xu and Rudall, 2006). The Annonaceae encompass many taxa with strong perianth differentiation. In Artabotrys hexapetalus L.f.

(Annonaceae), the sepals are much reduced in contrast to the petals illustrating the division of labour between bud protection and pollinator attraction (Fig. 1D).

How the angiosperm flower evolved is one of the great questions still unanswered by modern science. This task has been made difficult by the fact that plant lineages at the base of the angiosperm phylogenetic including Amborella and the Nymphaeaceae have flowers which are derived in several of their characters (Endress and Doyle, 2015; Sauquet et al., 2017). Similar difficulties have been encountered by authors attempting to understand the evolution of the core eudicot flower, a key innovation in the diversification of flowering plants. The pentamerous pentacyclic flowers of the core eudicots appear to have evolved rapidly, but how this most successful floral bauplan evolved is not known (Ronse De Craene, 2017).

Figure 1 Examples of perianth structure in the flowers of basal angiosperms. (A) Illicium henryi Diels (Schisandraceae), apical view of flower with undifferentiated perianth showing the transition from bract to petaloid tepals in a spiral. (B) Austrobaileya scandens White (Austrobaileyaceae), apical view of flower showing transition from bract to morphologically differentiated petaloid tepals in a spiral. (C) Magnolia grandiflora L. (Magnoliaceae), apical view of flower with weakly differentiated perianth arranged in three trimerous whorls. (D) Artabotrys hexapetalus L.f. (Annonaceae), lateral view of flower with perianth differentiated into one trimerous outer whorl of sepals (white arrow) and two inner trimerous petal whorls. Bract = B; P = petaloid tepal. Image credits: A, Scott Zona, NC State University, USA. Source: https://plants.ces.ncsu.edu [Accessed 02/08/2018]. B, Simon Goodwin, Twitter. Source: https://twitter.com/Gardenboi [Accessed -2/08/2018]. C, Missouri Botanic Garden. Source: http://www.missouribotanicalgarden.org [Accessed 02/08/2018]. D, Flowers of Sri Lanka, Twitter. Source: https://twitter.com/srilankanflora [Accessed 02/08/2018].

1.4. Evolution of the core eudicot perianth with emphasis on petals The core eudicots, of which there are approximately 260,000 species, or c.75% of extant angiosperm diversity (Zeng et al., 2017), have a bipartite perianth generally consisting of five sepals in the outer whorl and five petals alternating with the sepals in the inner (Endress, 1990). The advent of pentamerous flowers with a differentiated perianth is often regarded as a key innovation in angiosperm evolution, which has facilitated the generation of remarkable species diversity (Endress, 2010a). A general lack of perianth differentiation with distinctive petals in the basal angiosperms and basal eudicot grade, resulted in the widely accepted view that core eudicot petals, which form the inner whorl of a bipartite perianth, are andropetals derived from stamens (Endress, 1986). The belief that core eudicot petals are ubiquitously of androecial origin stems from the observation that fertile or sterile stamens (staminodes) in the basal angiosperms and basal eudicots such as the Ranunculales have varying degrees of elaboration and are involved in pollinator attraction (Endress, 1995; Ronse De Craene and Smets, 2001). In these groups the androecium clearly plays an important role in attracting pollinators. However, petaloid staminodes which cannot easily be distinguished from petals, but are identifiable by their position in the flower, occur in only a few eudicot taxa. For example, the outer antesepalous stamen whorl has been replaced by petaloid staminodes in all members of the family Theophrastaceae (Caris and Smets, 2004; Figure 2). Ronse De Craene (2007) argued that andropetals are in fact rare and have limited distribution in only a few taxa. If this is true, then the origin of the pentamerous pentacyclic flower and bipartite perianth typical of the core eudicots is a mystery.

Figure 2 Example of petaloid staminodes in the Theophrastaceae (Ericales): Jacquinia nervosa C.Presl, apical view of flower with whorl of large petaloid staminodes alternating with the petals. Petal = P; Staminode = St. Stamen = S. Image credit: Fabian A. Michelangeli, Source: www.diversityoflife.org [Accessed 05/08/2018].

The early diverging eudicots grade which includes the Ranunculales, Proteales, Trochodendrales and Buxales encompass significant floral diversity with strong links to that of the basal angiosperms (Ronse De Craene et al., 2003). Whilst taxa belonging to these groups generally have reduced dimerous flowers, several families in the Ranunculales exhibit remarkable floral diversity (Ronse De Craene et al., 2003). Within the morphologically diverse Ranunculaceae, there are taxa with dimerous, trimerous and pentamerous perianths. However, the occurrence of pentamerous flowers in the Ranunculaceae is not linked with the origin of the core eudicot pentamerous bipartite perianth but is a case of convergent evolution (Ronse De Craene, 2010).

It has been suggested by several authors that the ancestor of all eudicots had a dimerous flower, like many families within the early diverging eudicots and the Gunnerales, which is sister to all other core eudicots (Soltis et al., 2003; Doyle and Endress, 2011). But such assumptions

15 appear to have been made based primarily on the phylogenetic position of these basal taxa, with little consideration for the morphological transitions required. It has since been demonstrated using a comparative morphological approach that the reduced dimerous perianth found in the genus Gunnera (Gunneraceae), does not represent an ancestral state, but instead has evolved along with a suite of other specialised characters as part of a transition to wind pollination (Wanntorp and Ronse De Craene, 2005). Similar cases of floral reductions as adaptations to wind pollination can be found within the basal eudicot grade, i.e. Platanus (Proteales) (Wanntorp and Ronse De Craene, 2005). Researchers asking questions regarding the origin of the core eudicot bipartite perianth must instead look first to the lineages in the basal grades of both the rosids and asterids. These grades include the orders Berberidopsidales, Saxifragales, Santalales, Vitidales and Caryophyllales. Of particular interest are the genera Berberidopsis and Aextoxicon, which until linked by molecular evidence were not believed to be closely related (Nandi et al., 1998). They have now been placed in two families, Berberidopsidaceae and Aextoxicaceae in the order Berberidopsidales (Angiosperm Phylogeny Group, 2016).

Berberidopsis is unusual for the core-eudicots in that it has bisexual flowers with an undifferentiated spiral perianth which transitions from outer sepals to inner petals. Study of the floral ontogeny of Berberidopsis corallina Hook. f. revealed that organs arise along a divergence angle of 137.5°, the ‘golden’ angle, required for a shift between a spiral phyllotaxis and a whorled pentamerous configuration of organs with a 2/5 spiral initiation pattern (Ronse De Craene, 2004). Ronse De Craene therefore agues for a ‘predisposition for pentamery’ in the genus. In subsequent studies on other members of the Berberidopsidales, Ronse De Craene argued that species in the order each lie on a transitional gradient from spiral flowers without perianth differentiation to those with a whorled phyllotaxis and differentiated sepals and petals. This transition is believed to be driven by factors which directly affect the influence of spatial pressure acting on the flower throughout its development. For example, recent work on Berberidopsis beckleri (F. Muell.) Veldkamp revealed that there is a less condensed arrangement of flowers on inflorescences, a greater number of bracts, and greater variability in the number of perianth organs and stamens in flowers compared to B. corallina (Ronse De Craene, 2017). There is also no evidence of change in plastochron, the time interval between the initiation of subsequent organs, between outer and inner perianth parts. A change in plastrochron is recognised as fundamental for a transition from acyclic spiral flowers to those with a whorled arrangement.

Interestingly, there is greater differentiation of inner and outer tepal morphology in B. beckleri compared to B. corallina (Ronse De Craene, 2017). Whilst in Berberidopsis there is a transitional series between outer ‘sepals’ and inner ‘petals’ as in many basal angiosperms (Fig. 1), in Aextoxicon there is a distinction between an outer sepal whorl and inner petal whorl, as in the rest of the core eudicots (Ronse De Craene and Stuppy, 2010; Fig. 3). Yet there is little other similarity with other haplostemonous core eudicot flowers. The compaction of flowers on the inflorescences of B. corallina and Aextoxicon, in association with a flattening of the floral meristem and reduction in the number of organs, presents a scenario for how pentamerous diplostemonous flowers with a bipartite perianth may have evolved within the core eudicots (Ronse De Craene, 2017).

The transition from an undifferentiated perianth with spiral phyllotaxis typical of the basal angiosperms, to a biseriate pentamerous perianth may have occurred early in eudicot evolution through a gradual disruption of the plastochron in flowers with an organ divergence angle of 137.5°, leading to the arrangement of tepals in two whorls (Ronse De Craene, 2007; Ronse De Craene and Stuppy, 2010; Ronse De Craene, 2017). Such a transition is more likely on a structural basis than the derivation of a bipartite pentamerous perianth from two dimerous whorls of tepals (Endress and Doyle, 2009; Ronse De Craene and Brockington, 2013).

Figure 3 Images highlighting variation in the perianth of members of the Berberidopsidales (A) Flowers of Berberidopsis beckleri (Berberidopsidaceae) has a perianth with spiral phyllotaxis with transitional organ identity similar to that of basal angiosperms (B) Aextoxicon punctatum (Aextoxicaceae) has a differentiated pentamerous (occasionally hexamerous) perianth arranged in two whorls, five outer sepals and five inner petals. Image credit: A, Arven Pepiniere. Source: www.arven-boutique.com [Accessed 11/08/2018]. B, Wikimedia Commons. Source: https://commons.wikimedia.org [Accessed 11/08/2018].

Based on this model of perianth evolution, bracteopetals are ancestral for all core eudicots. This is strongly supported by the observation that the petals of most core eudicots are more sepal-like than stamen-like (Takhtajan, 1991). Only a few core eudicot taxa possess andropetals, which have been acquired through an evolutionary process of petal loss in association with pollination mechanism, and a subsequent need to ‘reinvent’ attractive petals (Ronse De Craene and Brockington, 2013). It is therefore important that future works on the evolution of the eudicot flower recognise the distinction between true bracteopetals and andropetals. This distinction is central to our understanding of the evolutionary history of all major eudicot groups.

1.5. Genetic control of floral organ identity Of central importance to the study of floral morphology is the distinction between positional homology of floral organs, which reflects the evolutionary history of flowers, and organ identity, which is the product of gene expression regimes acting on different organ positions within the flower and can produce homeotic changes (Kirchoff, 2000; Ronse De Craene, 2003).

The so-called ABC model of floral organ identity was suggested by Coen and Meyerowitz in 1991 to explain the floral developmental genetics which control floral organ identity in the model species Arabidopsis thaliana (Brassicaceae) and Antirrhinum majus (Plantaginaceae). Under this model, the identity of floral organs, which must be arranged in the evolutionarily conserved whorled floral bauplan of the core eudicots, is controlled by temporal activation of three key gene sets in specific regions of the floral meristem during development (Coen and Meyerowitz, 1991). A-class genes are expressed in the outermost whorl. The expression of APETALA1 (AP1) with one of the four SEPALLATA genes (SEP1-SEP4) determines sepal identity. The B-class MADS-box genes PISTILLATA (PI) and APETALA3 (AP3) (and their synonymous homologs) are expressed in the second whorl of the differentiated core eudicot perianth at the same time as the A-class genes, thus determining petal identity. Subsequently, stamen cell identity is determined when the C-class gene AGAMOUS (AG) is turned on and expressed in combination with AP3, PI and either SEP1,2 or 3. Carpel cells are specified when only AG and either SEP1,2 or 3 are expressed (Alvarez-Bullya et al., 2010; Sablowski, 2010). Experimentation with Arabidopsis mutants has demonstrated that not only are these combinations necessary for the development of the different organs within flowers, but the resulting gene products are sufficient to artificially transform leaves into floral organs outside of flowers (Honma and Goto, 2001; Pelaz et al., 2001). Conversely, homeotic replacement of sepals and petals with leaves occurs in Arabidopsis APETALA mutants (Bowman et al., 1991). Not only do these experiments substantiate the ideas pioneered by Goethe some 230 years ago that sepals, petals, stamens and carpels are all the result of ‘morphogenesis’ of the same basic organ type, the leaf. They also reveal that the use of genetic data alone cannot answer questions regarding the homology of floral organs between plants in different taxonomic groups and at different taxonomic levels.

Since the ABC system governing organ identity has been observed in several distantly related taxa within the core eudicots, it has been suggested that a petal identity program regulated by B-class MADS-box genes must have arisen early in angiosperm evolution (Jaramillo and Kramer, 2007). However, it is unsurprising that floral organ identity genetics in basal angiosperm taxa do not work in the same manner as in the core eudicots. Many basal angiosperm flowers have a gradual transition from outer sepaloid tepals to inner petaloid tepals within the perianth. This has been observed in many basal angiosperms including all families belong to the order Austrobaileyales and in Amborella trichopoda Baill. (Amborellaceae), sister to all other extant angiosperms (Endress, 2011). This transitional gradient of organ types

19 is indicative of overlapping expression of floral organ identity genes from the periphery to the centre of a floral meristem. This form of gene expression has been appropriately named ‘fading borders’, with Amborella as a representative model system (Buzgo et al., 2004). Under this model, weak and overlapping organ identity gene expression occurs in floral meristem regions where organs appear to be transitionary in nature. In particular, B-class genes, which are expressed in petals and stamens under the ABC model, have a gradually fading influence on organ identity toward the periphery of the floral meristem, giving one ‘set’ of organs characteristics commonly associated with a different set (Buzgo et al., 2004).

In light of current knowledge of floral organ identity genetics, determining whether petal-like organs are homologous in divergent or even closely related angiosperm taxa is therefore complicated, especially when developmental data is lacking. Throughout the evolutionary history of any given angiosperm lineage, perianth organs may become much reduced, almost lost then regained, or be reinvented entirely in response to complex interactions with the environment and pollinators within it (Ronse De Craene and Brockington, 2013). From a narrow focused developmental genetics standpoint, any floral organs which express petal-like features such as colourful pigmentation, could be misinterpreted as petals based solely on gene expression within them. However, sepals, bracts, staminodes and true petals can all be petal- like in appearance. Therefore, gene expression data alone is insufficient to explain the processes involved in perianth evolution within the angiosperms. Floral organs observed in different taxa may be incorrectly determined to be homologous, or of independent origin based on this data. To understand the angiosperm perianth, and indeed all organs associated with flowers, comparative morphology offers a powerful tool to make inference on the homologies of floral organs between taxa, and regarding floral evolution generally.

1.6. An introduction to Croton

1.6.1. Diversity and Ecology Croton L. (Euphorbiaceae) is one of the largest angiosperm genera comprising over 1200 species (Govaerts et al., 2000; Frodin, 2004). Whilst most Croton are woody shrubs or trees, there are several herbaceous annuals, lianas and creeping species (Arévalo et al., 2017). Croton species are recognised as an ecologically important component of xeric and mesic floras in many regions around the world, e.g., South and Central America, Africa and Madagascar,

20 northern Australia, continental Asia and the Malaysian archipelago (Berry et al., 2005). In particular, some Croton species are known to be adapted to thrive in disturbed habitats, with significant seedling recruitment following anthropogenic disturbance, natural tree fall, or fire (Ruthven and Synatzke, 2002; Tesfaye et al., 2008). In dry tropical forests in the north-eastern interior of Brazil, which have been greatly disturbed and modified by cattle farming and agriculture, Croton species have been identified as among the most important pioneer plant species during habitat regeneration (Gomes et al., 2012). In a dry shrubby community in Ecuador, the pioneer species Croton wagneri has been identified as an important ‘nurse plant’, which act as so-called community hubs, resulting in greater community species richness and plant coverage within C. wagneri patches (Espinosa et al., 2014).

The pollination ecology of Croton species has not been well documented. In fact, the literature appears extremely contradictory on the issue. For example, several instances of wind pollination have been reported in the genus (Dominguez and Bullock, 1989; Bullock, 1994). In the case of C. suberosus it has been argued that wind is the primary pollen vector (Dominguez and Bullock, 1989) and that the nectar produced within flowers functions to attract plant defenders such as predatory wasps, not pollinating insects (Dominguez et al., 1989). These are extremely controversial claims since the nectaries are located directly adjacent to pollen covered stamens within flowers. A subsequent study on the same species rejected these ideas using pollinator exclusion experiments which show that insect visitation is essential for successful reproduction (Narbona and Dirzo, 2010). Noted, is that flowers were visited by almost four times more wasps than bees. This reveals an interesting interaction in which predatory wasps not only act as a pollinator, but also defend the plant from herbivores. First impressions can be misinterpreted with regard to pollination systems in plant groups such as Croton which have many small flowers close together on a single inflorescence.

Of note is the diversity of extrafloral nectaries present in many Croton species as in other members of the family. These have been implicated in maintaining mutualisms with ants which protect the plant from herbivores (DeVries and Baker, 1989).

1.6.2. Morphology There are several vegetative characteristics of the genus which makes their identification in the field relatively straightforward. These include a pungent odour, watery latex (clear or coloured), presence of stellate or lepidote trichomes, often petiolar glands, and orange

21 senescent leaves (Van Ee et al., 2011). The Euphorbiaceae as a group are well known for great diversity in their inflorescences and in floral structure.

This diversity reflects the complex evolutionary history of the family. As the second largest genus in the family after Euphorbia, there is particular diversity in the flowers of Croton species (Gagliardi et al., 2017). The inflorescences of Croton are generally indeterminate racemes (floral unit as solitary flowers) or thyres (floral unit as a cyme) (Claßen-Bockhoff and Bull-Hereñu, 2013) with unisexual flowers. Flowers are always unisexual, and plants are mostly monoecious. Within inflorescences, male flowers are generally located toward the apex and female flowers at the base (Caruzo and Cordeiro, 2007). There is a distinctly different perianth in male and female flowers of most species (Webster, 1967). Male flowers have a valvate or imbricate calyx comprised of five sepals and five petals alternating with the calyx lobes. Female flowers share a calyx of five sepals, which can be valvate or imbricate, however in most species petals are absent or there are filamentous structures in the same position as the petals of male flowers. This is true in all but two New World sections of the family, both of which have well developed petals in pistillate flowers, viz. section Alabamenses and section Eluteria, and in some Old World taxa such as C.grattissimus Burch., which have not yet been taxonomically placed (Brown et al., 1925; Aplet et al., 1994; Webster, 2005). These filamentous structures have been variously interpreted in the past as reduced petals, glands and staminodes (De-Paula et al., 2011; Caruzo and Cordeiro, 2013; Gagliardi et al., 2017). However, to date no study has compared the development of these filamentous structures petals with those which are well-developed in the taxa mentioned. Similar structures in the position of petals have been recorded in the female flowers of several other genera within the Euphorbiaceae including Hevea, Codiaeum, Microcacca, Acalypha and Mercurialis. Similarly, their identity has been variously interpreted in each of these groups (De Paula et al., 2011).

The flowers of both Croton and sister genus Astraea have variously lobed extrastaminal nectaries located in the position between the petals, or opposite the sepals. These variously fuse in both male and female flowers and can form an entire ring at the base of the gynoecium in female flowers (Caruzo and Cordeiro, 2007). Caruzo and Cordeiro (2007) interpreted the nectaries of Croton as of receptacular origin. However, this assumption is not based on ontogenetic observation. Croton nectaries have also been described as staminodes, due to their position between the androecium and perianth and resemblance to stamens (De-Paula et al., 2011; Gagliardi et al., 2017). This interpretation is despite their late initiation in the floral

22 ontogeny, after all floral organs. Initiation very late in the development is characteristic of a receptacular origin of structures such as nectaries, coronas and nectar spurs (Ronse De Craene, 2010; Claßen-Bockhoff and Meyer, 2015). The interpretation of De-Paula et al. (2011) therefore rests primarily on anatomical data including the observation that the nectaries are vascularised by divergences of the sepal traces. The use of vascular data alone to infer homologies is outdated and should not be the basis for such conclusions. Several studies have shown that organ vascularisation is not strongly evolutionarily conserved, and that the development of vascular bundles depends on local auxin accumulation and tissue growth patterns (Prusinkiewicz and Runions, 2012). Just as auxin is involved in the regulation of floral phyllotaxis, it is also central to determining the growth of vascular bundles toward and into the floral organs during development (Reinhardt et al., 2003). Based on this evidence, it is important not to overinterpret the orientation of vascular bundles, especially in structures which develop late in the floral ontogeny as their development is based on all number of forces acting on the floral meristem.

The androecium within the genus is extremely variable. Reported stamen numbers range from five to over one hundred (Webster, 1993). A synapomorphy of the genus is that the tip of staminal filaments are inflexed or curved inwards in bud until anthesis (Berry et al., 2005). Very little work has been published on the evolution and development of the androecium in the genus. Two recent developmental studies have shown that stamens may arise centripetally or centrifugally on a convex receptacle, and that the number of stamens in each whorl is dependent on available space on the meristem. On occasion a single stamen initiates in the floral centre in position of the gynoecium (De-Paula et al., 2011; Gagliardi et al., 2017).

Croton species have a tri-carpellate, tri-locular gynoecium with one ovule on axile placentation per locule. This morphology is extremely well conserved throughout the Euphorbiaceae (Ronse De Craene, 2010; Endress et al., 2013).

1.6.3.Taxonomy As one of the most speciose genera, Croton has a complex taxonomic history. Traditionally the genus has been included within the Euphorbiaceae based on many shared morphological characters with Euphorbia, notably the distinctive gynoecium. Molecular phylogenetics has confirmed the taxonomic position of Croton within the Euphorbiaceae (Angiosperm Phylogeny Group, 2016). Within the Euphorbiaceae, Croton is circumscribed within the subfamily Crotonoideae (Berry et al., 2015). Within the subfamily, there have been many proposed tribes

23 and genera. However, in the Crotoneae, the proposed tribe which includes Croton, the various circumscriptions have been shown to be inaccurate using molecular phylogenetic evidence (Berry et al., 2015). Webster (1993) described 40 sections within tribe Crotoneae, each with a single species representative. However, little inference was made as to how each of these sections relate to each other. Recent molecular work with extensive species sampling has resulted in a phylogeny with the respective positions of sections of tribe Crotoneae as proposed by Webster (1993) (Berry et al., 2005). Three genera, Astraea, Basilocroton, Acidocroton and Ophellantha are positioned as sister to Croton within the tribe. Webster (1993) included Astraea as a section of Croton due to shared morphological characters, particularly inflexed anthers in bud and similar trichome morphology.

To date, work on the molecular phylogenetics of Croton has been entirely centred on New World taxa. In light of molecular phylogenetic work conducted by Berry et al. (2005) and subsequently by Van Ee et al. (2011), taxonomic revisions are underway within the sections themselves (Caruzo and Cordeiro, 2013). Work will likely continue to be focused on New World taxa for the foreseeable future.

1.7. Research Aims The aim of this study is to observe and compare the floral ontogeny of three Croton species each with different floral morphologies. At maturity, there are differences is the shape of the floral meristem, the perianth, nectaries, number and arrangement of stamens and in the morphology of the gynoecium amongst others. Two of the species, C. alabamensis Smith ex Chapman and C. schiedeanus Schltdl., both have well-developed petals in male and female flowers, a condition atypical of the genus as a whole. The third, C. chilensis, is more typical of the genus at large in that there are well-developed petals in the male flowers only. In C. chilensis, and in the majority of Croton species, it is unknown if the filamentous petals in female flowers are homologous to male petals (Van Ee et al., 2011). Given that C.alabamensis (section Alabamenses) and C.schiedeanus (section Eluteria) are not closely related according to the most recent phylogenetic analysis of the genus conducted by van Ee et al. (2011), I propose that there are two most probable evolutionary scenarios regarding the petals in female flowers. The first is that petal development in female flowers is suppressed by some unknown genetic means in the majority of Croton species, giving rise to the typical filamentous petal. Under this scenario, this genetic control must be switched off in section’s Alabamenses and

Eluteria. This is instinctively the most parsimonious explanation as it does not require multiple ‘reinventions’ of petals in disparate lineages within the Croton phylogeny.

An alternative hypothesis is that Croton petals are not bracteopetals, but are instead andropetals derived from sterile stamens. This is the view expressed by Gagliardi et al. (2017). It is even plausible that the strongly developed petals of male flowers are not homologous to the filamentous petals typical of female flowers. Under such a scenario, the petals of female C.alabamensis and C.schiedeanus represent the endpoint of a stamen-staminode-petal transition which is at various stages of progression throughout the genus. However, there is no doubt that such a system would require a complex genetic architecture to be maintained and seems unlikely in an evolutionary context.

Since only a few ontogenetic studies have been performed in the genus, none of which have included sections Alabamenses or Eluteria, such hypotheses have not yet been tested using a powerful comparative floral development approach. Through detailed observation and construction of an ontogenetic series for both male and female flowers of the three species mentioned, this study will answer the following questions. Are Croton petals of tepaline origin like the sepals, or are they derived from sterilised and transformed stamens? Are the petals in female C. alabamensis and C.schiedeanus homologous to those in male flowers? What are the implications for the evolutionary identity of the filamentous petals found in C. chilensis and throughout the genus at large?

In addition to answering the above questions, comparing the floral development and morphology of these three species, which are dispersed throughout the Croton phylogeny, is a powerful tool to understand the evolution of many aspects of floral evolution within the genus. As such, the development of the androecium and the morphology of nectaries will be compared and discussed. Scanning electron microscopy will be used to to answer these questions in conjunction with comparative anatomy using light microscopy.

2. Methods This comparative study was conducted using spirit material of Croton schiedeanus, C. alabamensis and C. chilensis which had been fixed in FAA solution (90% ethanol at 70%, 5% acetic acid, 5% formaldehyde at 40%) then preserved in 70% ethanol (Table 1).

Table 1 Origins of spirit material used in this study.

Species Codes Collector with Place of collection/Origin number/ Accession number C. alabamensis E. A. PFA8 Murdock and Pratt's Ferry, Cahaba, Smith Berry PFA8 Alabama, USA. Material used for study was garden grown at Murdock garden, Beltsville, Maryland, USA. C. schiedeanus Schltdl. 865 LA Ronse De Craene Belize C. schiedeanus Schltdl. 1093 LA Stoddart 02 Belize C. chilensis Mull. 20090241A 20090241A RBGE/Chile

2.1. Floral Dissection, Critical Point Drying and preparation for Scanning Electron Microscopy

For morphological and ontogenetic study using a Scanning Electron Microscope (SEM), floral material was prepared by removing bracts and bracteoles tending flowers of various developmental stages under a compound light microscope. The calyx of developing flowers was also removed to allow viewing of petal, androecium and gynoecium development. As trichomes arise early in the development of inflorescences of the taxa studied, these were also removed where possible.

Dissected flowers were dehydrated using a graded ethanol (EtOH) series. Specimens were placed in 95% EtOH for ten minutes, followed by five minutes in 100% EtOH. They were then transferred to 100% acetone dried with a molecular sieve for five minutes before being exchanged to a second vial for a further five minutes.

Dehydrated samples were transferred to a K850 Critical Point Dryer (CPD) pre-cooled to approximately 4°C using liquid carbon dioxide (CO2). The CPD specimen chamber was flooded with liquid CO2 and the stirrer was turned on for two minutes. The exhaust valve was opened slowly to release the liquid, ensuring the meniscus never fell below the marked level and that the specimen is always covered . The chamber was then flooded as before, and the

27 stirrer turned on for two minutes. This process of liquid exchanges was repeated a total of ten times. Following ten liquid CO2 exchanges, the chamber was once again flooded to the level marked on the machine. The heater was turned on and the temperature and pressure allowed to rise to the range of 35-39°C and 1100 psi respectively. At approximately 30°C and 1100 psi, the critical point was reached. The pressure was slowly released from the chamber. Dried specimens were immediately removed to a jar containing silica crystals pre-dried in a heat oven at 40°C for one hour.

Dried specimens were mounted on 12.5mm diameter aluminium SEM pin stubs with aluminium cored carbon tabs. Mounted specimens were coated with platinum in an Emitech K575X sputter coater then viewed using a Leo Supra 55-VP SEM.

2.2. Tissue clearing and viewing

For examination of perianth vasculature, floral tissue was cleared by immersion in 10% sodium hydroxide (NaOH) solution for up to three weeks until the tissue became transparent. Samples were rinsed in three changes of distilled water before being bleached by immersion in 2% sodium hypochlorite for ten minutes, or until the flower turned white. Samples were rinsed in three changes of distilled water then left in the final change for at least ten minutes. Flowers were dehydrated in a graded ethanol series (50%, 70% and 95%) under vacuum for ten minutes per alcohol concentration. They were then stained in 1% safranin solution in 95% ethanol for one hour before being rinsed in 95% ethanol for at least ten minutes. Samples were de-stained in 95% ethanol acidified with three drops of 37% HCL for ten minutes. Flowers were rinsed in two changes of 100% ethanol then being stored for one week in 100% ethanol. Cleared and stained flowers were observed using a dissecting microscope (Zeiss stemi 2000-C) and dissected organs were photographed with an AxioCam MRc 5 (Zeiss).

3. Results

3.1. Organography of C. alabamensis

Flowers are spirally arranged on many-flowered terminal racemes with few female flowers toward the base and many male flowers further up the axis. Female flowers mature first followed by males in sequence toward the apex of the inflorescence (Fig. 4A). Male flowers were observed to occasionally vary in their merism, with tetramerous and hexamerous flowers observed alongside the normal pentamerous condition. All flowers are subtended by a bract and two lateral bracteoles. Both have a fimbriate margin and are covered in lepidote trichomes on their outer surface.

A B

Figure 4 Reproductive structures of Croton alabamensis. (A) Inflorescence showing few mature female flowers at the base (Black arrow). Male flowers mature in spiral sequence toward the apex (White arrow). (B) Floral diagram of a male flower. (C) Floral diagram of a female flower. (Star = Floral axis; Black arcs with spine = bract/bracteole; Black arcs = sepals; White arcs = Petals; Grey lobes = nectaries; = stamen; = Trilocular gynoecium with single carpel on axile placentation per locule). Dashed lines represent the limits of a cup shaped

30 hypanthium. Image A credit: Virginia Polytechnic Institute and State University, USA. Source: www. http://dendro.cnre.vt.edu [Accessed 07/06/2018]. Male and female flowers both have a pentamerous bipartite perianth consisting of well- developed sepals and petals each with a fimbriate margin of stellate trichomes, and an outer surface covered with lepidote trichomes (Fig. 4A,B; Fig. 5A). Sepals have a broad base and appear fused at their bases through development of a hypanthium. Petals have a broad base and are free. A cup shaped hypanthium is evident in the mature flowers of both sexes. The petals are inserted on the rim of the hypanthium. Five large bilobed nectaries occur inside the petal whorl in antesepalous position in both sexes (Fig. 4A,B). These are flattened and fused into a ring at their bases in male flowers but are free in female flowers (Fig. 5B,F). They are covered in stomata in both sexes, particularly on the outer surface (Fig. 5E). The androecium in male flowers is arranged in three whorls each with five stamens (Fig. 4B). The stamens are inserted on the slope of the hypanthium and are curved toward the centre in bud. The filaments are long with a slightly swollen base with few stellate trichomes (Fig. 5C). There are two laterally inserted anthers which dehisce longitudinally (Fig. 5D). The connective is narrow. There is no trace of a reduced gynoecium in male flowers. Similarly, there is no trace of staminodes in female flowers. In female flowers, the globular tricarpellate ovary consists of three locules, each containing a single pendant, epitropous ovule on axile placentation (Fig. 5G). There are three long and broad bifid styles which curve downward at maturity. Each has an extensive stigmatic slit running over the length on the upper surface. The surface of the ovary is covered with many overlapping lepidote trichomes (Fig. 5G). A single anomalous flower with a bicarpellate gynoecium was observed.

A B

P N

N P S S N N P N

C D E

F G G N

P N

S Ov

Figure 5 Morphological structures of mature male and female flowers of Croton alabamensis. (A-E) Male flowers; (F-G) Female flowers. (A), Detail of petal and sepal of male flower at anthesis; both organs share a common morphology each with lepidote trichomes on the outer surface (black arrows) and stellate trichomes covering the margin (white arrows). (B), Dissected mature male flower with five bilobed nectaries opposite the sepals; they appear fused into a ring at the base; the central androecium has been entirely removed. (C), Lateral view of mature stamen; note the simple trichomes emerging near the base. (D), Detail of anther and pollen sacs with narrow connective. (E), Stoma from the upper surface of a nectary in mature male flower. (F), View of bilobed nectary of a mature female flower; petals and sepals have been removed. (G), Gynoecium of mature female flower showing a single pendent, epitropous ovule on axile placentation; outer and inner integuments are visible. S, sepal; P, petal; N,

32 nectary; G, Gynoecium; Ov, ovule. Scale bars: (A) = 60 μm; (B) = 300 μm; (C, F) = 200 μm; (D) = 40 μm; (E) = 2 μm; (G) = 400 μm. 3.2. Organogeny in male flowers of C. alabamensis The sequence of organ development was investigated in male flowers. Flowers mature in a spiral sequence towards the apex of the inflorescence. The floral meristem arises within the protection of a bract which initiates and attains substantial size before any floral organs initiate. Two bracteoles develop in lateral position either side of the floral meristem (Fig. 6A). The first sepal initiates in the position furthest from the axis on a convex meristem. Sepal initiation then follows a 2/5 spiral sequence (Fig. 6B). The first sepal to initiate is large and almost covers the entire bud during development. Following the initiation of all five sepals, the floral meristem begins to regress vertically; this leads to the development of a cup shaped hypanthium (Fig. 6C-E). Five broad petal primordia initiate alternating with the sepals at the beginning of hypanthial development (Fig. 6D). A rapid spiral initiation is likely, however more intermediate stages are required to confirm this. Petal development is unidirectional, with those on the abaxial side of the flower developing more rapidly than the others (Fig. 6E-I). Following initiation of all five petals, a basically centripetal stamen initiation follows. Stamens initiate in two whorls of five, with the antepetalous stamen primordia developing more rapidly in early stages (Fig. 6E-G). Stamen development then briefly follows the same unidirectional pattern as the petals as a third whorl of five stamens is initiated toward the floral centre (Fig. 6H-J) resulting in fifteen stamens arranged in three whorls of five on the edges of the hypanthial cup within the protection of the differentiating petals (Fig. 6L-N). Five large bilobed nectaries develop inside the petal whorl in antesepalous position late in the floral ontogeny. Unfortunately, their development was difficult to record due to the abundance of trichomes covering the floral meristem and the bases of all organs. Prior to anthesis the stamens are curved inward, and the sepal, petal and floral centre are covered in many stellate trichomes (Fig. 6O). The five sepals appear to be fused at their bases at maturity, however this appearance is due to the extensive growth of the hypanthium throughout the floral development.

A B S2 C S2 Br S4 S4

S1 S2 B S1 S5 S1 S5

Br S3 S3

S3 D S1 E F P1 P P1 P S1 P * * S5 S3 S4

P P * * P P S2 S4 * S5 P S2 P

G H P P I P P1 P A2 P A3 P A1 P A P P P A A S1 P1 P P S1 P1

J K L P1 P1 A2 A1 P A1 P P P A3 A2 A3 P P P P P P1 P

O M N P P P P S S

P P P P P P S A3 A1 P P P P P A2

Figure 6 Sequence of floral development of male flowers of Croton alabamensis. (A) Apical view of initiation of first and second sepals on a convex meristem; Bract and bracteoles were removed. (B) Young sepal primordia show a 2/5 initiation spiral pattern; sepal one and three have been removed. (C) Development of a cup shaped hypanthium prior to petal initiation;

Sepals one to four have been removed. (D) Initiation of five petals on the edge of the hypanthium; sepals have been removed. (E) Initiation of two whorls of five stamens in the hypanthial cup; Note the antepetalous stamen primordia are slightly more developed (asterisks). (F) Development of stamens in the hypanthium. (G) Similar stage showing unidirectional development of petals. (H) Initiation of a third stamen whorl toward the centre of the hypanthium; Note the unidirectional development of petals. (I) Differentiation of petals as trichomes begin developing on the margin; Note unidirectional development of petals and stamens; Numbers indicate different stamen whorls. (J) Older bud with three petals on one side clearly larger than the two opposite; Three stamen whorls formed. (K) Older bud showing growth of the petals and stamens; There is no trace of carpel primordia at the centre of hypanthium. (L) Older bud with several trichomes developing on the margin of larger petals (arrow). (M) Older bud with petals enclosing the inner flower; lepidote trichomes beginning to develop on the outer surface (arrow); The largest petal has been removed. (N) Older bud with development of petals. (O) Apical view of flower nearing anthesis; stamens are arranged in three whorls of five and are curved toward the centre; Note petals are larger than sepals. B, bract; Br, bracteole; S, sepal; P, petal; A, stamen. Numbers give the order of organ initiation. Scale bars: (A-I) = 20 μm; (J-M) = 60 μm; (N) = 40 μm; (O) = 400 μm.

3.3. Organogeny in female flowers of C. alabamensis The sequence of organ development was investigated in female flowers. Flowers mature in a spiral sequence from the base to approximately the middle of the inflorescence where there is a transition to male flowers (Fig. 4A). Like male flowers, the floral meristem arises within the protection of a bract which initiates and attains substantial size before the any floral organs initiate. Two bracteoles develop in lateral position either side of the floral meristem. Sepal initiation could not be recorded in female flowers, however a 2/5 spiral initiation is implied based on the imbricate aestivation at later stages of development. Like male flowers, a cup shaped hypanthium begins to develop prior to petal initiation (Fig.7A). Five broad based petal primordia initiate between the sepals. Carpel initiation with congenital fusion appears to occur rapidly, whilst the petals are still in early stages of development (Fig. 7A). There are no traces of staminodes or a reduced androecium within the female flower. Petal development is unidirectional, with those on the abaxial side of the flower larger than others (Fig. 7B-C). The tricarpellate gynoecium develops rapidly in the floral centre (Fig. 7B-F). A bilobed style develops on the top of each carpel (Fig. 7D-F). Many lepidote trichomes develop on the outer surface of the ovary (Fig. 7D-H). At maturity, the petals of female flowers are large with a morphology identical to those of male flowers (Fig. 7H). Five large bilobed nectaries develop inside the petal whorl at the base of the gynoecium in antesepalous position late in the floral ontogeny. Unfortunately, their development was difficult to record due to the abundance of trichomes covering the floral meristem and limited material. Like in male flowers, the five

35 sepals appear to be fused at their bases at maturity, however this appearance is due to the extensive growth of the hypanthium throughout the floral development.

P P A S B P C P G P P P G B P G P P P P P P P

E F D P P P P P

P P P P P

G H

S S S P S P

Figure 7 Sequence of floral development of female flowers of Croton alabamensis. (A) Apical view of young flower showing initiation of petal and carpel primordia; Four sepals have been removed (B) Development of petals and tricarpellate gynoecium in a cup shaped hypanthium; Note simple trichomes on the margin of largest petals and unidirectional development of the petals. (C) Development and differentiation of petals. (D) Older bud showing petal and carpel development. (E) Abnormal bud with bicarpellate gynoecium; Note lepidote trichomes developing on the outer surface of petals. (F) Initiation of development of bilobed styles; Developing petals enclosing the gynoecium. (G) Elongation of the bilobed styles; Note the abundance of lepidote trichomes on the outer surface of sepals and petals. (H) Flower nearing maturity with well-developed gynoecium. S, sepal; P, petal; G, gynoecium. Scale bars: (A-D) = 20 μm; (E-G) = 30 μm; (H-J) = 60 μm; (K) = 100 μm; (L) = 200 μm.

3.4. Perianth vasculature of male and female flowers of C. alabamensis An anatomical investigation was conducted on sepals and petals of both sexes. The morphology and vasculature of the perianth organs are indistinguishable between male and female flowers (Fig. 8A-D). The sepals of both sexes have a single primary vascular bundle which gives rise to secondary and tertiary veins on both sides of the organ (Fig. 8A,C). The petals of both sexes have a single vascular bundle from the base to the tip. In male and female flower this gives rise to two secondary lateral veins approximately one quarter of the way along the axis (Fig. 8B,D). Several other lateral veins then branch off the primary vascular bundle on both sides further up the organ’s axis.

Figure 8 Floral anatomical structures of Croton alabamensis. (A)A cleared sepal from male flower with one main vascular bundle (arrow) which branches along the axis of the organ; the attached nectary is not vascularised. (B) A cleared petal from male flower with single vascular bundle which branches into two lateral veins a quarter of the way along the axis (arrow). (C) Cleared sepal from female flower with one vascular bundle branching along the axis of the organ (arrow). (D) Cleared petal from female flower with single vascular bundle which branches into two lateral veins a quarter of the way along the axis (arrow). Scale bars: (A-D) = 200 μm.

3.5. Organography of C. schiedeanus Many flowered racemes are located at branch tips and in the leaf axils. Flowers are spirally arranged with a single or few female flowers at the base and many male flowers towards the aapex. Female flowers mature first followed by males in spiral sequence toward the inflorescence apex (Fig. 9). Immature female flowers can be identified by their pedicel, which is distinctly robust compared to that of male flowers (Fig. 9). Male and female flowers are subtended by a bract and two lateral bracteoles. Both have a fimbriate margin and are covered in lepidote trichomes on their outer surface. Male flowers were observed to occasionally vary in their merism, with tetramerous flowers observed alongside the normal pentamerous condition.

A B

Figure 9 Reproductive structures of Croton schiedeanus. (A) Inflorescence showing few, stalked mature female flowers at the base (arrow). Male flowers mature in spiral sequence toward the apex. (B) Floral diagram of a male flower. (C) Floral diagram of a female flower. For key to floral diagram symbols see Figure 4 legend. Image A credit: Reinaldo Aguilar (Flickr). Source: https://www.flickr.com [Accessed 07/07/2018].

Male and female flowers both have a pentamerous bipartite perianth consisting of well- developed sepals and petals with a fimbriate margin of simple trichomes, and an outer surface sparsely covered with lepidote trichomes (Fig. 9B,C; Fig. 10A, B). Sepals have a broad base and appear fused at their bases through development of a hypanthium. Petals are broadly clawed and free with quincuncial aestivation (Fig. 10A). The floral meristem is convex. Large nectaries are prominent in flowers of both sex. In males there are five free dorsally flattened, bilobed nectaries inside the petal whorl in antesepalous position (Fig. 10B,E). The nectary in female flowers is a ring encircling the base of the gynoecium. However, this ring is not independent of the petals which arise from common tissue (Fig. 10F). No stomata were observed on the nectaries of either sex. In male flowers with abnormal merism, the same number of nectaries alternates with the petals. The androecium in male flowers is basically obdiplostemonous with ten stamens arranged in two whorls. There is a single eleventh stamen located at the floral centre (Fig. 9B). Stamens are inflexed in bud (Fig. 10D). The filaments are long and straight and are lacking trichomes at the base (Fig. 10C). There are two laterally inserted anthers which dehisce longitudinally (Fig. 10D). The connective is narrow. There is no trace of a reduced gynoecium in male flowers as the single stamen occupies this position. Similarly, there is no trace of staminodes in the female flowers. The globular tricarpellate ovary consists of three locules, each containing a single pendant, epitropous ovule on axile placentation (Fig. 10G). There are three long and broad styles which curve downward at maturity. Each has an extensive stigmatic slit running over the whole length on the upper surface. The surface of the ovary is covered with many overlapping lepidote trichomes.

1A B 1 P P S

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P G Ov Ov S P N P Figure 10 Morphological structures of mature male and female flowers of Croton schiedeanus. (A-E) Male flowers; (F-G) Female flower. (A), Male flower showing quincuncial petal aestivation prior to anthesis. (B), Mature male flower with five bifid nectaries, each opposite a sepal. (C), Lateral view of mature stamen. (D), Detail of dehiscent anther. (E), Close view of nectary position in mature male flower. (F), View of nectary in female flower which is comprised of a complete ring at the base of the gynecium; the closest petals have been removed. (G), Dissected gynoecium with ovules, one per carpel; each is attached via an apical placenta (arrow). S, sepal; P, petal; N, nectary; G, Gynoecium; Ov, ovule. Scale bars: (A-C) = 300 μm; (D) = 80 μm; (E) = 90 μm; (F) = 100 μm; (G) = 200 μm.

3.6. Organogeny in male flowers of C. schiedeanus The sequence of organ development was investigated in male flowers. Male flowers mature in a spiral sequence from the first, which is inserted above the female flowers, to the apex of the inflorescence (Fig. 9A). The floral meristem arises within the protection of a bract which initiates and attains substantial size before any floral organs initiate. Two bracteoles develop in lateral position either side of the floral meristem. The first sepal initiates in the position furthest from the axis on a convex meristem. Sepal initiation then follows a 2/5 clockwise spiral sequence (Fig. 11A-B). The first sepal to initiate is large and almost covers the entire bud during development. Five young petal primordia appear to expand into available space between the developing sepals (Fig. 11B-C). A rapid spiral initiation is likely, however more intermediate stages are required to confirm this. Five broad petal primordia develop and differentiate rapidly along a unidirectional gradient, with those furthest from the inflorescence axis developing more rapidly than the others (Fig. 11D-E). This unequal growth of the petals continues throughout the development of the flower (Fig. 11G-K). Prior to anthesis, the petals remain unequal in size (Fig.11N). Following initiation of all five petals, a centrifugal stamen initiation follows. A basically obdiplostemonous androecium initiates in two whorls of five stamens. The antisepalous stamen primordia, located near the floral apex initiate first (Fig. 11E), followed by five stamens opposite the petals (Fig. 11F-G). A single stamen then initiates at the floral centre in the position of the gynoecium (Fig. 11H-J). At later stages of development five large flattened and slightly bilobed necatries develop inside the petal whorl in antesepalous position late in the floral ontogeny. The stamens curve inward during late development (Fig. 11L-N). The petals develop a fimbriate margin of simple trichomes and lepidote trichomes on the outer surface, like the sepals (Fig. 11K-O). The five sepals appear to be fused at their bases at maturity, however this appearance is due to the extensive growth of the hypanthium throughout the floral development.

S A B S2 C P P S2 P P S S5 S S5 S4 P S4 P P P S3 P S1 S3 S S1 S P B

D S4 E F P P P * * P S2 * A1 P P * P * * P A1 * S1 P P * * P P P * S5 P P S3

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K P L S J P P P A2 A1 A1 S A2 P P P P A3 P P P P P P P

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Figure 11 Sequence of floral development of male flowers of Croton schiedeanus. (A) Apical view of convex meristem; Sepals were removed. (B) Five petal primordia initiating; The size of dissected sepals indicates a 2/5 initiation spiral pattern. (C) Initiation of petal growth; Sepals have been removed. (D) Unidirectional development of petals; Note that the meristem has been damaged. (E) Initiation of a whorl of five stamens opposite the sepals (asterisks). (F) Initiation

42 of a second whorl of five stamens opposite the petals (asterisks); Stamen initiation is centrifugal on a convex meristem; Note the primordium at the centre of the flower (arrow). (G) Evident unidirectional development of petals and stamens. (H) Two stamen whorls visible; Development of central primordium is delayed (arrow). (I) Development and differentiation of petals with unidirectional development; Initiation of a single stamen in the centre of the flower (arrow). (J) Older bud showing the growth of petals and stamens. (K) Older bud with differentiation of petals; Trichomes developing on the margin of larger petals. (L) Older bud showing arrangement of the androecium; A single stamen has been removed. (M) Older bud with petals enclosing the inner flower; lepidote trichomes beginning to develop on outer petal surface; stamens are curved toward the centre and becoming inflexed. (N) Apical view of flower nearing anthesis; (O) Lateral view of flower at anthesis. B, bract; Br, bracteole; S, sepal; P, petal; A, stamen. Numbers give the order of organ initiation. Scale bars: (A-J) = 20 μm; (K) = 40 μm; (L-N) = 200 μm; (O) = 400 μm.

3.7. Organogeny in female flowers of C. schiedeanus The sequence of organ development was investigated in female flowers. One to three female flowers mature near the base of the inflorescence (Fig. 9A). Female flowers develop very early in the growth of the inflorescence when it is entirely concealed by multiple inflorescence bracts. Like male flowers, the floral meristem arises within the protection of a bract which initiates and attains substantial size before the any floral organs initiate. Two bracteoles develop in lateral position either side of the floral meristem. Sepal initiation follows a 2/5 spiral initiation pattern in an anticlockwise direction (Fig. 12A-B). Five broad petal primordia initiate between the sepals (Fig. 12C-F). There are no traces of staminodes or a reduced androecium within the female flower. Petal development appears unidirectional, with those on the abaxial side of the flower slightly larger than others (Fig. 12G). The tricarpellate gynoecium develops rapidly in the floral centre (Fig. 12G-K). The gynoecium develops three branched styles and a covering of many overlapping lepidote trichomes throughout the later stages of development (Fig. 12J- K). At maturity, the petals of female flowers are large with a morphology identical to those of male flowers (Fig. 12L). Five large bilobed nectaries develop and fuse to form a ring inside the petal whorl at the base of the gynoecium in antesepalous position late in the floral ontogeny (Fig. 12J). Late in development, the pedicel of female flowers elongates substantially giving the appearance of a stalked flower at maturity.

S2 A B C S P S S5Scale bars: A-I = 10umBr, J-MS1 = 30um, N= 20um, O =200um P S4 B S2 S3 P Br S3 S Br P P S S1 S

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P P1 S5 P4 P S1 P B P P3 P2 S P P5 S4 P S2

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P N P S Figure 12 Sequence of floral development of female flowers of Croton schiedeanus. (A) View of young flower; Sepal one has been removed (B) Young sepal primordia show a 2/5 spiral initiation pattern in an anticlockwise direction; The bract has been removed. (C) Initiation of petal primordia; Four sepals have been removed. (D) Similar stage showing petal primordia; Sepals have been removed. (E) Growth of petal primordia; Note that the meristem has been slightly damaged. (F) Young petal primordia show a 2/5 spiral initiation pattern; Three sepals have been removed. (G) Development of petals and initiation of the gynoecium. (H) Older bud showing petal and gynoecium development; Note the trichomes appearing on the petal margins. (I) Petal and carpel elongation; Three petals have been removed. (J) Development of a nectary at the base of the gynoecium. (K) Older bud with developing petals and gynoecium; Note the lepidote trichomes gynoecium surface. (L) Older bud with well-developed petals enclosing the gynoecium; Note the simple trichomes on the petal margin (black arrow), and lepidote trichomes on the outer surface (white arrow). B, bract; Br, bracteole; S, sepal; P, petal. Numbers give the order of organ initiation. Scale bars: (A-D) = 20 μm; (E-G) = 30 μm; (H-J) = 60 μm; (K) = 100 μm; (L) = 200 μm.

3.8. Perianth vasculature of male and female flowers of C. schiedeanus An anatomical investigation was conducted on sepals and petals. Unfortunately, the sepals of male flowers were not able to be studied due to damage during preparation. The sepals of female flowers have three vascular bundles entering the organ at the base. Each of these branches further up the axis giving rise to several secondary lateral veins (Fig. 13B). The petals of male and female flowers have a single primary vascular trace which branches into three slightly above the base. These then give rise to many tertiary veins (Fig. 13A,C).

Figure 13 Floral anatomical structures of Croton schiedeanus. (A) A cleared petal from male flower with one vascular bundle which branches into three slightly above the base (arrow). (B) Cleared sepal from female flower with single vascular bundle which branches into three slightly above the base of the organ (arrow). (C) A cleared petal from female flower with one primary vascular bundle branching along the axis of the organ (arrow); other veins which do not originate from this primary bundle are also visible. Scale bars: (A-C) = 200 μm.

3.9. Organography of C. chilensis Many flowered racemes are located on shoot apices and in leaf axils. Flowers are spirally arranged with many female flowers at the base and many male flowers above these to the inflorescence apex. Female flowers mature first followed by males in sequence towards the apex of the inflorescence (Fig. 14A). Female and male flowers are easily identified by long protruding styles or stamens respectively. Flowers are subtended by a bract and two lateral bracteoles which are covered in many stellate trichomes.

Male flowers have a pentamerous bipartite perianth consisting of well-developed sepals and petals (Fig. 14B). Sepals have a broad base and appear fused at their bases in both sexes. The margin and outer surface of sepals are covered with many intertwining simple trichomes (Fig.). The petals of male flowers have a broad apex and slightly clawed base. Aestivation is cochleate prior to anthesis (Fig. 15A). Male petals have many simple trichomes at the base (Fig. 15B).

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Figure 14 Reproductive structures of Croton chilensis. (A) Inflorescence showing many mature female flowers at the base and many mature and immature male flowers towards the apex. (B) Floral diagram of a male flower. (C) Floral diagram of a female flower. For key to floral diagram symbols see Figure 4 legend. Image A credit: Jardin Botanico Nacional (Viña del Mar), Chile. Source: https://www.flickr.com [ Accessed 07/07/2018].

The petals of female flowers are much reduced (Fig. 14C). Rarely is more than one petal developed in mature flowers. These have a filamentous appearance and are very narrow in comparison to male petals (Fig. 14C) and the apex often bears trichomes. The floral meristem is convex. Nectaries are prominent in flowers of both sex. In males there are ten free globular nectaries inside the petal whorl, two at the base of each petal (Fig. 15B,E). The nectary in female flowers is a ring encircling the base of the gynoecium. This appears to be composed of five fused bilobed nectaries each in antesepalous position (Fig. 15F). No stomata were observed on the nectaries of either sex. The androecium in male flowers is arranged in five whorls on the convex meristem with either twenty-four or twenty-five stamens (Fig. 14B; Fig. 16L). Stamens are inflexed and curved inwards in bud. The filaments are long and straight with many intertwined trichomes at the base (Fig. 15C). There are two laterally inserted anthers which dehisce longitudinally (Fig. 15D). The connective is narrow. There is no trace of a reduced gynoecium in male flowers; stamens occupy the entire floral meristematic apex. Similarly, there is no trace of staminodes in the female flowers. The globular tricarpellate ovary consists of three locules, each containing a single pendant, epitropous ovule on axile placentation (Fig. 15G). There are three styles which branch at half their length and curve erratically at maturity (Fig. 17I). Each has an extensive stigmatic slit running over the whole length on the upper surface. The surface of the ovary is covered with many intertwining stellate trichomes.

A B 1 P P P S S P N N P P S P S P P P S

C D E 3 4 5

P P N N N N

6F 7G G N P N Ov S P S

Figure 15 Morphological structures of mature male and female flowers of Croton chilensis. (A-E) Male flower; (F-G) Female flower. (A), Male flower showing cochleate petal aestivation prior to anthesis. (B), Mature male flower with ten nectaries appearing as pairs at the base of each petal. (C), Lateral view of mature stamen. (D), Detail of dehiscent anther with pollen grains (arrow). (E), Close view of nectary position in mature male flower. (F), View of nectaries in female flower forming a complete ring at the base of the gynoecium; undeveloped petals are labelled; sepals have been removed. (G), Ovule and apical placenta (arrow) in mature gynoecium. S, sepal; P, petal; N, nectary; G, Gynoecium; Ov, ovule. Scale bars: (A) = 60 μm; (B) = 1000 μm; (C) = 300 μm; (D) = 30 μm; (E) = 200 μm; (F) = 40 μm; (G) = 100 μm.

3.10. Organogeny in male flowers of C. chilensis The sequence of organ development was investigated in male flowers. Male flowers mature in a spiral sequence from the first, which is inserted above the female flowers, to the apex of the inflorescence. The floral meristem arises within the protection of a bract which initiates and attains substantial size before the any floral organs initiate (Fig. 16A). Two bracteoles develop in lateral position either side of the floral meristem. The first sepal initiates in the position furthest from the axis on a convex meristem. Sepal initiation then follows a 2/5 clockwise spiral sequence (Fig. 16B). The first sepal to initiate is large and almost covers the entire bud during development. Five petal primordia initiate alternating with the developing sepals (Fig. 16C). A rapid spiral initiation is likely, however more intermediate stages are required to confirm this. Five broad petal primordia develop and differentiate rapidly along a unidirectional gradient, with those furthest from the inflorescence axis developing more rapidly than the others (Fig. 16D-E). Like in both C. alabamensis and C. schiedeanus, this unequal growth of the petals continues throughout the early development of the flower (Fig. 16F-I). Following initiation of all five petals, a centripetal androecium development follows, beginning with the initiation of five stamens opposite the sepals which develop to occupy the space between the petals (Fig. 16E-H). A second stamen whorl initiates opposite the petals. This is obscured from view by the developing petals which begin to ‘bend’ due to increasing spatial pressure imposed by the expanding floral meristem (Fig. 16G-H). Stamen primordia then initiate over the remainder of the meristematic surface almost simultaneously (Fig. 16H-J). As the flower matures these appear loosely arranged in a third, fourth and fifth whorl (Fig. 16L). However, due to the spatial pressure imposed by adjacent primordia, the position of the stamens is slightly different in each flower. The final stamen to initiate is always forced into the floral centre giving the false impression of a central stamen as in C. schiedeanus (Fig. 16L-M). Ten small globular nectaries develop inside the petal whorl, two at the base of each petal, late in the floral ontogeny. Stamens curve inward during late development and become inflexed (Fig. 16N). Petals are larger than sepals at maturity, have an imbricate aestivation and lack trichomes except at the base (Fig. 15A,B). The five sepals appear to be fused at their bases at maturity, however this appearance is due to growth of the receptacle throughout the floral development.

S4 S2 A B C B S2 S4 S1 S5 P P

B S5 P S1 S3 S3 B

D E F P P P P P P P * * P P * P * P P

P G P H P I P P A1 P A5 A3 P P A5 P A4 A3 A4 S A3 A1 A1 A1 P P A2 P S A1 A1 P S S P S S

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P A5 A5 A5 P A4 P A2 A4 A3 A3 A4 A3 P A2 A1 A2 A1 P A1 P P P S M N O P S P P P S P P

P P S P S P P P S P Figure 16 Sequence of floral development of male flowers of Croton chilensis. (A) View of young inflorescence showing convex floral meristems; Bracts were removed. (B) Young sepal primordia show a 2/5 initiation spiral pattern; sepal two is damaged. (C) Initiation of petal primordia; Sepal one has been removed. (D) Development of petal primordia; Sepals have been removed. (E) Initiation of antesepalous stamens (asterisks); Note the meristem has been damaged. (F) Development of petals and stamen primordia (G) Initiation of a second and third

50 stamen whorl; Note stamen primordia directly opposite the petals are mostly obscured from view (H) Initiation of a fourth and fifth stamen whorl toward the centre of the meristem. (I) Stamen primordia are visible at five different levels; Clearly defined whorls are not yet visible. (J) Older bud with developing stamens; Stamen whorls appear chaotic. (K) Older bud showing growth of the stamens; Five stamen whorls are visible; To petals have been removed. (L) Older bud showing arrangement of the androecium on the convex meristem; Twenty-four stamens are visible arranged in five whorls. (M) Older floral bud; Stamen whorls become less easily defined as the organs mature; Note that the final stamen to initiate is forced into the centre due to space limitation. (N) Older bud nearing anthesis. (O) Apical view of flower at anthesis. B, bract; Br, bracteole; S, sepal; P, petal; A, stamen. Numbers give the order of organ initiation. Scale bars: (A-E) = 20 μm; (F-I) = 40 μm; (J-L) = 60 μm; (M) = 200 μm; (N) = 400 μm; (O) = 600 μm.

3.11. Organogeny in female flowers of C. chilensis The sequence of organ development was investigated in female flowers. Flowers mature rapidly in a spiral sequence from the base to approximately the middle of the inflorescence where there is a transition to male flowers (Fig. 14A). An abundance of intertwining stellate trichomes cover the entire inflorescence making observation of early stages difficult. The floral meristem arises within the protection of a bract which initiates and attains substantial size before any floral organs initiate. Two bracteoles develop in lateral position either side of the floral meristem. Sepal initiation was unable to be recorded in female flowers, however a 2/5 spiral initiation as in male flowers is implied based on an imbricate aestivation at later stages of development. Five broad petal primordia initiate between the sepals. One is clearly larger than the others implying that initiation is not simultaneous (Fig. 17A-B). Carpel initiation with congenital fusion appears to occur rapidly, whilst the petals are still in early stages of development. The initiating gynoecium occupies all available space inside the petal whorl (Fig. 17A). As the gynoecium develops, petal development is arrested (Fig. 17B-E). Five bilobed necatries fused into a ring develop at the base of the gynoecium (Fig. 17C-E). As the gynoecium matures three styles develop which branch into two half way along their length. The gynoecium becomes covered in many stellate trichomes. The petals begin to elongate, perhaps following a 2/5 spiral sequence (Fig. 17F). The filamentous petals continue to elongate whilst the bud remains closed (Fig. 17F-H). At maturity when the sepals open to expose the floral centre, some petals appear to continue developing laterally giving rise to an entirely petal-like organ (Fig. 17I). The five sepals appear to be fused at their bases at maturity, however this appearance is due to the growth of the receptacle throughout the floral development.

S P A P B C S S P P P P S P N N P P P S P S

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P P P N N N Figure 17 Sequence of floral development of female flowers of Croton chilensis. (A) View of young flower showing initiation of petal and carpel primordia; Four sepals have been removed (B) Development of petals and tricarpellate gynoecium. (C) Retarded development of petals relative to gynoecium; Bilobed nectaries developing at base of carpels, opposite sepals; Sepals have been removed. (D) Older bud showing carpel development. (E) Older bud showing development of three bilobed styles; Note lepidote trichomes developing on the outer surface of gynoecium; Nectaries are well developed. (F) Elongation of some petals; Note that they are filamentous in appearance. (G) Development of a petal; Note the abundance of lepidote trichomes and simple trichomes on the gynoecium surface. (H) Contrast between one well developed petal and one with retarded growth (arrow). (I) Mature flower with a single well- developed petal visible. S, sepal; P, petal; G, gynoecium; N, nectary Scale bars: (A-D) = 20 μm; (E) = 60 μm; (F-H) = 100 μm; (I) = 400 μm.

3.12. Perianth vasculature of male and female flowers of C. chilensis An anatomical investigation was conducted on sepals and petals of both sexes. Anatomically the perianth organs are very different between male and female flowers (Fig. 18A-D). The sepals of both sexes have a single primary vascular trace. In male flowers, this branches giving rise to lateral veins on both sides along the axis of the organ (Fig. 18A). In female flowers, the primary vascular bundle branches into three slightly above the base (Fig. 18C). The petals of

52 both sexes have a single vascular bundle from the base to the tip. In male flowers this gives rise to two secondary lateral veins approximately one third of the way along the axis (Fig. B). In female flowers, the primary branches into several smaller veins along the length of the axis (Fig. D).

Figure 18 Floral anatomical structures of Croton chilensis. (A) Adaxial surface of a cleared sepal from male flower with one main vascular bundle (arrow) which branches along the axis of the organ. (B) A cleared petal from male flower with single vascular bundle which branches into two lateral veins a third of the way along the axis (arrow). (C) A cleared sepal from female flower with one main vascular bundle branching into three slightly above the base (arrow). (D) A cleared petal-like filamentous structure from female flower with single vascular bundle which branches into few smaller lateral veins along the axis of the organ. Scale bars: (A,C,D) = 200 μm; (B) = 400 μm.

4. Discussion 4.1. Petal evolution in Croton The majority of described Croton species lack well-developed petals in female flowers. However, two major new world sections, Alabamenses and Eluteria, contain species with equally well-developed petals in both male and female flowers. The species within these two sections are not closely related according to the most recent molecular phylogenetic analysis of the genus (Van Ee et al., 2011). This raises the question of whether their perianth morphology represents a case of convergent reinvention of petals in female flowers, or whether petals have been systematically reduced or lost throughout the evolution of the genus. A comparative morphological approach and study of the floral ontogeny of several species of interest, is a powerful tool for answering questions which molecular phylogenetics cannot, such as the most likely scenario of perianth evolution in Croton.

The results of this study show that the petals in male flowers of all three species analysed are characteristic of bracteopetals and are not of secondary staminodial origin. Many characteristics of the male floral ontogeny support this conclusion. These include a rapid initiation of five broad petal primordia, quickly following the elongation of the fifth sepal (Fig. 6 Fig. 11; Fig. 16). For contrast, petals of staminodial origin have a delayed initiation and primordia are characteristically narrow (Ronse De Craene, 2008). All five petals appear to initiate simultaneously, however their imbricate petal aestivation at maturity is indicative of a tepaline 2/5 spiral initiation pattern (Ronse De Craene, 2007). Additionally, petal growth is rapid, following a unidirectional pattern of development which gives rise to large petals of unequal size throughout the floral ontogeny. The morphological and anatomical data also support a tepaline origin of the male petals. Particularly in C. alabamensis, male petals are difficult to distinguish from the sepals at maturity. Both share a fimbriate margin of simple trichomes and a covering of many lepidote trichomes on the outer surface (Fig. 5A). They also share a broad base and each have one primary vascular bundle (Fig. 8A,B). Of note, is the difference in morphology at maturity between C. chilensis male sepals and petals. Whilst the sepals are broad based with a single primary vascular trace, the petals are clawed at maturity (Fig. 18A,B). Clawed petals have previously been discussed as a derived character common within the rosids (Ronse De Craene and Brockington, 2013).

Since this is the first study to catalogue the development of flowers in the New World sections Alabamenses and Eluteria, until now it has remained unknown whether the well-developed

55 petals of female flowers are homologous to those in male flowers. Similarly, the nature of the reduced filamentous petals found in the female flowers of species throughout the genus has been obscured. In both C. alabamensis and C. schiedeanus, the morphology and vasculature of mature female petals appears identical to those of male flowers in every respect (Fig. 8B,D; Fig. 13A,C). Likewise, the ontogeny of the perianth in C. schiedeanus is indistinguishable between male and female flowers (Fig. 11; Fig. 12). Unfortunately, the earliest developmental stages of female C. alabamensis floral development were not available for SEM observation. However, petal primordia in female flowers share the same position, morphology, rapid unidirectional sequence of development and unequal size during ontogeny, as those in male flowers (Fig. 6; Fig. 7). There is little doubt that the male and female petals are homologous in both species.

This data raises questions regarding the evolution of the perianth within the genus as a whole. In the third species studied, C. chilensis, there is a significant contrast in morphology between the petals of male and female flowers. Based on the floral ontogeny presented here, it is clear that the petals of male C. chilensis are also bracteopetals of tepaline origin, as evidenced by the characteristics already described for the other two species studied (Fig. 16). Previously the filamentous petals characteristic of most female Croton flowers, including have been variously interpreted. Based on morphological and anatomical studies, they have been interpreted as reduced petals or glands (Nair and Abraham, 1962; Webster, 1993; Freitas et al., 2001; Caruzo and Cordeiro, 2007; Riina and Berry, 2010). More recently, using an ontogenetic approach led De-Paula et al. to conclude they were indeed reduced petals, whereas Gagliardi et al. interpreted the structures as staminodes (De-Paula et al., 2011; Gagliardi et al., 2017).

The ontogenetic data presented here reveal that the broad petal primordia in female C. chilensis flowers initiate much like those observed in male flowers (Fig. 17A-B). However, their development is subsequently arrested, as the large gynoecium undergoes development. Of particular interest is that the young petals are unequal in size, as in C. alabamensis and C. shiedeanus (Fig. 7B; Fig. 12G; Fig. 17B). This is indicative of a 2/5 spiral sequence of initiation. Later in the development, the reduced petals of female C. chilensis then begin to elongate. At this point the carpels already occupy all space within the floral centre. Due to great spatial pressure imposed by the gynoecium during elongation, the mature petals are long and narrow at maturity. Whilst observation of the petal elongation was difficult due to the size of the gynoecium and abundance of trichomes, this process may follow a 2/5 spiral sequence (Fig. 17E-I). Although this is speculation and requires further observation. In addition, at least one

56 larger petal does occasionally occur in the female flowers of C. chilensis (Fig. 17I). Whilst well-developed petals do not usually occur in the species, the genetic potential to produce such structures remains.

Together, this data indicates that the filamentous petals in female C. chilensis flowers are indeed true bracteopetals. It corroborates the conclusions reached by De-Paula et al. (2011) in their ontogenetic study of several Croton species, and directly refutes the conclusions of Gagliardi et al. (2017) that female filamentous petals of Croton are staminodes and part of a vestigial androecium.

Based on these observations and on those of the other respective authors mentioned, it is apparent that the petals of female Croton flowers have undergone a process of repeated reductions throughout the evolution of the genus. To me, the floral ontogeny of C. chilensis suggests that there is a genetic control which delays petal development shortly after organ initiation. In this species, petal growth then resumes late in the floral ontogeny, by which time there is little space remaining for their full development. A subtle change in the rate of petal development has resulted in fixed evolutionary change and diversification within the female perianth. Such a process, referred to as heterochrony, was first introduced by Haeckel (Haeckel, 1875). Heterochrony has since been recognised as one of the major drivers of morphological evolution (Li and Johnston, 2000; Ronse De Craene, 2018). Takhatajan discussed how plant morphological diversity, particularly floral diversity, has likely been generated through processes of subtle developmental modifications, deletions and inventions through evolutionary time. He demonstrated that all floral organs evolved from leaf-like structures via changes in the timing, rate and location of developmental events (Takhtajan, 1972; Takhtajan, 1991). As discussed by Li and Johnston (2000), genes and the systems which regulate their expression are responsible for the timing of developmental processes. Alteration in the timing of developmental processes must therefore occur because of changes in the timing of gene- expression (Li and Johnston, 2000).

Time, size and pressure are the three crucial factors which govern the development of floral organs and resulting floral morphology (Ronse De Craene, 2018). Even small changes in the timing of an organs’ initiation will inevitably alter its relationship with adjacent structures. A delay of organ initiation or development may therefore lead to reduction or loss because of increased spatial pressures imposed by other more rapidly developing organs. Intuitively, this can have great consequences in flowers with a strictly whorled arrangement as in the

Pentapetalae, where different whorls have organs whose identity is determined by different genes (Alvarez-Bullya, 2010) and which develop independently from one another (Ronse De Craene, 2018).

I propose that the developmental variation in the perianth of Croton species presents an interesting model system for the study of floral evolution, and for testing theories put forward by respected floral morphologists. The variable perianth of female Croton flowers is evidence that ancestrally inherited phenotypes are not always expressed clearly in all members of a clade. Said character would therefore not be regarded as synapomorphic using a traditional cladistics approach. However, all members of a clade may have the genetic potential to express that character. This has been referred to as deep morphology (Scotland, 2010). Morphological characters (here petals in female flowers) which are present in a clade (here Croton) but not all its members have been referred to as ‘apomorphic tendencies’ by Endress (2003) and ‘cryptic apomorphies’ by Ronse De Craene (2010). Such characters are extremely useful for understanding the evolution of a clade. However, they are largely ignored, or misinterpreted as convergent evolution by taxonomists using a traditional cladistics approach. Future work on the Croton perianth should consider and explore these concepts.

Finally, the variably reduced female perianth of Croton species presents an opportunity to study the interaction of two systems governing floral morphology, the genetic system and the temporal-spatial system of development studied by floral morphologists. If the petals of female C. chilensis flowers which have a delayed development had enough space, would they fully develop, or remain filamentous at maturity? In other words, do floral organs have a predetermined ultimate size, or is size governed purely by spatial constraints in the developing bud? This could be tested by genetic manipulation of the genes controlling carpel development (C-class genes). If the C-class gene AGAMOUS is not turned on during floral development, both stamens and carpels will fail to form (Sablowski, 2010). The absence of a large gynoecium in the floral centre of female Croton flowers would present an interesting case to explore the temporal-spatial hypotheses of floral development put forward by Li and Johnston (2000) and modified by Ronse De Craene (2018).

4.1. The identity of nectaries in Croton Nectaries which initiate late in the floral development are prominent in all three species studied (Fig. 5; Fig. 10; Fig. 15). At maturity these are generally five in number, bilobed, and located inside the petal whorl in antesepalous position. However, in the male flowers of C. chilensis, there are ten free globular nectaries appearing as pairs near the base of each petal (Fig. 15B,E). Throughout the genus, floral nectaries are variously fused, often into a complete ring at the base of the gynoecium (Freitas et al., 2001; Caruzo and Cordeiro, 2007; De-Paula et al., 2011). Caruzo and Cordeiro (2007) interpreted them as receptacular outgrowths, whereas both De- Paula et al. (2011) and Gagliardi et al. (2017) interpreted these nectaries as transformed staminodes. Given the late development of the nectaries in Croton, why De-Paula et al. (2011) did not interpret them as of receptacular origin remains unclear. Their interpretation and assumptions involving floral vasculature is discussed in detail in section 1.6.2. of this work.

Throughout the order Malpighiales, to which Croton and the Euphorbiaceae belong, hypanthial growth and well developed nectaries, occasionally forming a disk, are common (Ronse De Craene, 2010). A deep hypanthial cup with a nectary ring is present in members of the Chrysobalanaceae (Matthews and Endress, 2008). Developmental work suggests that the well- developed ring and flap-like nectaries of members of the Clusiaceae are receptacular in nature (Sweeney, 2008). The formation of the receptacular corona of Passiflora is a remarkable example of how novel structures can be generated from receptacular outgrowths late in the development of flowers (Claßen-Bockhoff and Meyer, 2016). Nectaries which have been variously interpreted are common within the Euphorbiaceae in genera closely related to Croton (Ronse De Craene, 2010). I suggest that the variable number (within a single ‘whorl’), position, fusion and late onset development of nectaries observed in C. alabamensis, C. schiedeanus and C. chilensis all point to a receptacular origin for the nectaries of Croton.

4.1. Evolution of the androecium in Croton The development of the androecium was highly variable between the three species studied. Stamen initiation is centripetal in both C. alabamensis and C. chilensis (Fig. 6; Fig. 16), with the outermost whorl of stamens opposite the sepals in both species (Fig. 4A; Fig. 14A). This pattern of development follows the temporal-spatial patterns of organ initiation common in most angiosperms, where the outer organs are initiated first and the last formed are those at the floral centre (Rudall, 2010; Brockington et al., 2013). In contrast, the development of stamens in C. schiedeanus is centrifugal, that is the inner whorl of five antesepalous stamens initiate

59 before the outer whorl which are positioned opposite the petals (Fig. 11B-I). This configuration remains at maturity (Fig. 9B). This condition, known as obdiplostemony, has been linked to a disruption of the development of the two stamen whorls in a diplostemonous androecium (Ronse De Craene and Bull-Hereñu, 2016). Ronse De Craene and Bull-Hereñu (2016) described three developmental conditions leading to obdiplostemony. The stamen initiation of C. schiedeanus with centrifugal obdiplostemony is rare (Ronse De Craene and Smets, 1995; Ronse De Craene and Bull-Hereñu, 2016). Obdiplostemony is evident in several other previously studied Croton species including C. fuscescens (De-Paula et al., 2011; Gagliardi et al., 2017), C. lundianus and C. glandulosus (De-Paula et al., 2011). Whilst a sufficient ontogenetic series is lacking for these species, in the data presented stamen initiation appears to follow the same pattern as C. schiedeanus. That is the two outermost stamen whorls are basically obdiplostemonous with a centrifugal development. Subsequent stamen whorls then initiate after the outermost alternisepalous whorl.

There are few reported cases of centrifugal obdiplostemony. Bello et al. (2008) reported a case in the genus Suriana (Surianaceae). Another reported the condition in Bruguiera parviflora (Rhizophoraceae) (Juncosa and Tomlinson, 1987). In the case of Suriana, the carpels are in an antepetalous position, and the antepetalous stamens are reduced in size (Bello et al., 2007). Such a positioning of the carpels appears to be tightly linked to this form of stamen development. An extensive review of the literature revealed that all forms of obdiplostemony are almost exclusively related to an antepetalous position of carpels (Table 1 in Ronse De Craene and Bull-Hereñu, 2016). Endress (2010) argued that it is the position of carpels in antesepalous position which is the clearest indicator for diplostemony, and that the reverse condition, antepetalous carpels is a driver of obdiplostemony. In an earlier publication, the same author cited antepetalous carpels as mandatory in a definition of obdiplostemony (Bachelier and Endress, 2009). Whilst the position of carpels clearly has a strong influence on development of the androecium, that is obviously not the case in the unisexual flowers of Croton. Endress (2010b) has also stated that there are no substantiated cases of centrifugal initiation of stamens in obdiplostemonous flowers. To my knowledge, centrifugal obdiplostemony hasn’t previously been reported in unisexual flowers. Croton is therefore a special case deserving of much further attention.

6. Conclusions This study highlights several interesting and exciting dynamics governing floral development and evolution. Croton is one of the most species rich genera in the angiosperms. The developmental work presented here highlights that the flowers of the genus also present diverse ontogenies. The ontogenies of reduced female petals which occur throughout the genus adds to existing questions regarding which genes are responsible for timing the initiation of floral organs, and whether their growth is also regulated genetically or primarily by spatial constraints. It raises the question of whether plants have the capability to ‘mute’ gene activity, such as those governing petal development, in response to spatial or environmental pressures. This is a particularly interesting concept in the unisexual flowers of Croton, where male and female flowers with contrasting petal morphology develop on the same inflorescence and share an identical genetic base. An obdiplostemonous androecium, which develops centrifugally, was observed in C. schiedeanus. This highlights a special case of stamen development of particular interest to floral morphologists which may also occur in other members of the genus. What causes this pattern of stamen initiation remains unclear. Excitingly, this appears to be the only case of centrifugal obdiplostemony in a unisexual flower reported in the angiosperms. This investigation should act as a base for further work on the floral ontogeny of Croton flowers. The ontogenetic and morphological characters they express makes them an excellent model system for studying the physical, genetic and phytochemical processes which govern floral development and evolution.

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