Evolution of the angiosperm flower: news and views from Darwin until now
Imagine roses, tulips, lilies and orchids – we all feel quite familiar with flowers, don't we? Many will be quite surprised, therefore, to learn that the modes and mechanisms of the origin and early diversification of the flowering plants (angiosperms) is still one of the most hotly debated matters of evolutionary biology.
The angiosperms provide us, directly or indirectly, with the vast majority of human food (such as vegetables and fruits) as well as with a lot of other economically important products (e.g. use in the preparation of clothes, furniture and drugs), and they also have great aesthetic value as ornamental plants. In addition, the contributions of angiosperms to biodiversity in terrestrial ecosystems can hardly be over-estimated. No wonder, therefore, that botanists and evolutionary biologists made considerable efforts to clarify the origin and diversification of the flowering plants. The apparently ‘sudden’ origin and rapid early morphological radiation of the angiosperms, as revealed by the fossil record, already intrigued Charles Darwin, who considered it a ‘perplexing phenomenon’ and even an ‘abominable mystery’ (Crepet, 1998
Like most other real mysteries, explaining the early evolution of angiosperms proved difficult, despite considerable efforts during the last one and a half centuries (Frohlich, 2003
). Major reasons for this are a quite uninformative (and probably extremely incomplete) fossil record and the great morphological gap between the flowers of angiosperms and the reproductive structures of gymnosperms, angiosperms' closest relatives; gymnosperms comprise extant conifers (including well-known taxa such as spruce and pine species), gnetophytes, cycads, Ginkgo, and diverse extinct groups including Bennettitales, Cordaitales, Corystosperms and Glossopterids (Doyle, 1998
). The morphological gap between gymnosperms and angiosperms leads to problems with homology assignments between reproductive organs from flowering plants and their putative ancestors and thus hampers an understanding of the transition from gymnosperm reproductive cones to angiosperm flowers (Frohlich, 2003
Flowers are characteristic features of angiosperms, and their origin is a major aspect of the ‘abominable mystery’. Given the importance of flowers one wonders that botanists have difficulties to define both precisely and comprehensively what actually a flower is. Bateman et al. (2006)
have listed about a dozen of historical circumscriptions, and these authors as well as Baum and Hileman (2006)
came up with updated definitions of a flower. As a consensus of these contemporary attempts, one may define a flower as a determinate, compressed, bisexual reproductive axis composed of megasporangia (carpels), microsporangia (stamens) and a sterile perianth composed of at least one sterile laminar organ. This is obviously not a comprehensive definition, since there are numerous types of flowers that lack one or more of these organ types, probably due to secondary simplification (e.g. unisexual flowers with only stamens or carpels).
This leads to the question as to which of these features represent the key characteristics of angiosperm flowers (for a detailed discussion of the synapomorphies of the angiosperms, see Donoghue and Doyle, 1989
). The presence of carpels enclosing the ovules is generally considered an essential character that distinguishes typical angiosperm flowers from gymnosperms' reproductive cones (Crane et al., 1995
; Endress, 2001
; Stuessy, 2004
). Another key feature of angiosperm flowers is a trait termed hermaphroditism, or bisexuality, describing the fact that male and female reproductive organs are united in one structure (or secondarily separated, as in the unisexual flowers of monoecious and dioecious angiosperms), while they might be primarily separated in different structures in gymnosperms. Moreover, flowers have a compressed reproductive axis, while gymnosperms have elongate ones (Baum and Hileman, 2006
). The presence of a perianth surrounding the reproductive organs, often including attractive organs of petaloid appearance (petals or tepals) is considered yet another typical feature distinguishing angiosperm flowers from gymnosperm cones. Often double fertilization yielding a nutritive endosperm is also considered an important angiosperm feature (Stuessy, 2004
; Kramer and Jaramillo, 2005
). Obviously, a flower is not a simple characteristic, but a complex of innovations (Baum and Hileman, 2006
Almost all terrestrial environments are currently dominated by flowering plants. To what extent the evolution of the flower contributed to the evolutionary ‘success’ of the angiosperms is unknown, but there is good reason to assume that it was of quite some importance. In addition to allowing efficient pollination by animals, thereby permitting population densities lower than in gymnosperms without inbreeding depression and extinction (Regal, 1977
), flowers also ‘catalyse’ seed dispersal via the formation of fruits, also often employing animal vectors. Flowers also increase the potential for reproductive isolation, e.g. by changes in pollinator specificity or flowering time (Grant, 1971
The tempo and mode of morphological changes by which the angiosperm flower originated has remained enigmatic. Based on circumstantial evidence, several authors have argued that bisexuality may have been one of the first steps during flower origin (see below, and Theissen et al., 2002
; Theißen and Becker, 2004
; Baum and Hileman, 2006
). Due to a lack of informative fossils, the other steps are even more speculative. Stuessy (2004)
hypothesized that angiosperms may have evolved slowly from seed ferns in the Jurassic, beginning first with the carpel, followed later by double fertilization, and lastly by the appearance of flowers. This series of events may well have taken more than 100 million years to complete, before an ‘explosive’ evolutionary diversification may have set in when the final combination of the essential angiosperm features was achieved (Stuessy, 2004
). Ignoring the question of carpel origin, Baum and Hileman (2006)
proposed that evolution of a bisexual axis via a gynomonoecious intermediate was the first step during flower origin, followed by evolution of floral axis compression and determinacy as step two, evolution of a petaloid perianth by sterilization of the outer stamens in step three, and origin of the dimorphic perianth of core eudicots in step four. What makes this scenario especially intriguing is the fact that for each of these steps an underlying developmental genetic mechanism is suggested by Baum and Hileman (2006)
. Thus the scenario outlined by these authors can in principle be experimentally tested by comparative developmental genetic studies in gymnosperms and angiosperms. This, however, is much easier said than done, given the poor performance of extant gymnosperms as developmental genetic model systems; they are all woody species requiring lots of growth space and many years of vegetative development until they reach the reproductive stage.
Various aspects of the evolutionary origin of the angiosperm flower have been reviewed by several authors during recent years (Crepet, 1998
; Frohlich, 1999
, 2006; Frohlich and Parker, 2000
; Ma and dePamphilis, 2000
; Albert et al., 2002
; Theissen et al., 2002
; Stuessy, 2004
; Theißen and Becker, 2004
; Theissen, 2005a
; Theißen and Kaufmann, 2006
; Baum and Hileman, 2006
; Bateman et al., 2006
; Doyle, 2006
). With the accumulating knowledge about the molecular genetic basis of flower development in some model plants the proximate causes and molecular mechanisms of floral evolution have found increasing interest. Here we focus on an especially well-understood aspect of flowers from a developmental genetic point of view: organ identity. We outline how two leading models explaining the specification of floral organ identity during individual plant development are helping to better understand flower origin and diversification.
Evolution of seed plants and the ancestral flower of crown group angiosperms
Understanding the phylogeny of seed plants (spermatophytes), a clade comprising angiosperms + gymnosperms, is an important prerequisite for understanding the evolution of the reproductive structures of these taxa, including flowers. Much has been learned about the phylogeny of seed plants in recent years by the use of molecular markers, even though some critical aspects have remained unresolved and controversial (Bateman et al., 2006
; Frohlich, 2006
Extant gymnosperms are morphologically very diverse and hence have usually been considered being paraphyletic, with gnetophytes (Gnetum
) often regarded as the sister group of the angiosperms (Doyle, 1998
). It thus came as a surprise that the vast majority of studies using molecular markers found moderate to strong support for extant gymnosperms being monophyletic (see, for example, Chaw et al., 1997
; Soltis et al.
; Winter et al., 1999
; Bowe et al., 2000
; Frohlich and Parker, 2000
; reviewed by Frohlich, 2006
). Extant gymnosperms and angiosperms may thus be sister groups that, as also suggested by molecular evidence (see e.g. Goremykin et al., 1997
), separated roughly about 300 million years ago. These hypotheses, however, are not easy to reconcile with the fact that the most ancient reliable angiosperm fossils, i.e. those of Archaefructus
, are just about 130 million years old (Sun et al., 2002
; Friis et al., 2003
), because this means there would have been about 170 million years of evolution during which the lineage that led to extant angiosperms left no recognizable traces in the fossil record (Bateman et al., 2006
). Thus especially some (but not all) paleobotanists do not accept the molecular-derived hypotheses about seed plant phylogeny, and consider the issue as unresolved (for an overview, see Bateman et al., 2006
; Frohlich, 2006
In any case, the inability to unambiguously identify an (extinct or extant) group of gymnosperms as the closest relatives of the angiosperms implies that the gap between the two kinds of seed plants could not be bridged yet.
There is evidence that either Amborella trichopoda
, or a clade of Amborella
together with Nymphaeales (water lilies) represent the most basal angiosperms (see, for example, Qiu et al., 1999
; Soltis et al.
; Barkman et al., 2000
; Graham and Olmstead, 2000
; for a review see Kuzoff and Gasser, 2000
; Soltis and Soltis, 2003
; Soltis et al. 2005
). As the next branch, this clade or grade is then followed by Austrobaileyales, a monophyletic group uniting Schisandraceae (now also including Illiciaceae; Frohlich, 2006
), Trimeniaceae and Austrobaileyaceae. [The grade of probably most basal angiosperms was originally termed ‘ANITA’ (Qiu et al., 1999
), and has recently been named ‘ANA’ (Amborella
, Nymphaeales, Austrobaileyales) (Frohlich, 2006
Assuming that Amborella
, Nymphaeales and Austrobaileyales indeed represent the most basal angiosperms, the flowers at the base of crown group angiosperms can be reconstructed by consideration of floral structures within this grade. These ‘ancestral flowers’ (Frohlich, 2006
) were probably already hermaphroditic and had an undifferentiated perianth, in which organs were arranged in more than two cycles or a spiral. Given their completeness and complexity these flowers at the base of crown group angiosperms, hence probably represent already a quite late, rather than initial stage during the origin of the flower as we know it. However, flowers with differentiated sepals and petals probably evolved even later during the evolution of angiosperms (Albert et al., 1998
; Kuzoff and Gasser, 2000
; Ronse De Craene et al., 2003
; Soltis et al., 2005
In the framework of our current view on angiosperm phylogeny, the clade comprising all flowering plants above the basally diverging lineages is called the ‘euangiosperms’, including the magnoliids, monocots, Chloranthaceae and eudicots. The phylogenetic relationships of these groups are unresolved. The eudicots comprise a grade of successive branches, with Ranunculales, including Papaveraceae, as sister to all other eudicots, and a large clade of ‘core eudicots’ containing the majority of all angiosperm species, including Caryophyllales (to which Polygonaceae belong), Rosids (to which Brassicaceae belong), Asterids, and several additional lineages (). The major eudicot model plant thale cress (Arabidopsis thaliana
; henceforth termed Arabidopsis) belongs to the plant family Brassicaceae within the eurosids (APG II, 2003
; Soltis and Soltis, 2003
Fig. 1. On the evolution of the ABC system of floral organ specification. On the right, different versions of the ABC system are shown. The ABC models differ either by the expression domains of the ABC genes, or by the organs specified by the different gene combinations. (more ...)
From the ABC model to the quartet model of floral organ identity
Eudicots represent the largest group of flowering plants, comprising about 75 % of angiosperm species (Buzgo et al., 2005
). Eudicots have relatively standardized flowers that typically consist of four different classes of organs arranged in four (or more) whorls at the tip of a floral shoot. The first, outermost whorl usually consists of green sepals resembling vegetative leaves. The second whorl is composed of often relatively large, coloured and showy petals. The third whorl contains the stamens, i.e. the male reproductive organs which produce the pollen. Finally, the fourth, innermost whorl contains the carpels, i.e. the female reproductive organs, which are often fused and inside of which, after fertilization, the ovules develop into seeds.
The structures of mature sepals, petals, stamens and carpels differ dramatically. Nevertheless, each floral organ starts its development as a little bulge generated by anticlinal and periclinal divisions of undifferentiated cells of the floral meristem. Thus each cell in the developing floral organ primordium somehow ‘recognizes’ its position within the flower, and differentiates accordingly into a cell type that is appropriate for the specific organ.
Based on the study of homeotic mutants in which the identity of floral organs is changed, genetic models were developed that explain how the different floral organs acquire their specific ‘identities’ during development (reviewed by Theissen, 2001). In Arabidopsis, homeotic mutants come in three classes, A, B and C. Ideal class A mutants have carpels rather than sepals in the first whorl, and stamens instead of petals in the second whorl. Class B mutants have sepals rather than petals in the second whorl, and carpels replace stamens in the third whorl. Class C mutants develop petals rather than stamens in the third whorl, and sepals instead of carpels in the fourth whorl. In addition, the flowers of class C mutants grow indeterminately, i.e. there is continued production of mutant floral organs inside the 4th whorl; this is in contrast to wild-type flowers, whose development ceases after carpel formation.
The phenotypes of class A, B and C mutants proved extremely informative; they suggested that flower development is sculpted by homeotic selector genes (also termed ‘floral organ identity genes’). These genes act as major developmental switches that control the entire genetic programme required for the development of a particular organ. The activities and interactions of floral homeotic genes have been described in several quite similar models (Haughn and Somerville, 1988; Schwarz-Sommer et al., 1990
), of which one, the ‘ABC model’ (Coen and Meyerowitz, 1991
) became widely known (). This ‘classical ABC model’ maintains that organ identity in each floral whorl is determined by a unique combination of three organ identity gene activities, called A, B and C. Expression of class A genes alone specifies the formation of sepals. The combination AB specifies the development of petals, and the combination BC specifies stamen formation. Expression of C alone determines the development of carpels. In order to explain the three classes of floral homeotic mutants, the ABC model proposes that the class A and class C genes negatively regulate each other, so that the class C gene activity becomes expressed throughout the flower when the class A gene function is compromised, and vice versa (for reviews of the ABC model, see Weigel and Meyerowitz, 1994
; Theißen, 2001
In Arabidopsis, class A genes are represented by APETALA1
) and APETALA2
), class B genes by APETALA3
) and PISTILLATA
), and class C genes by AGAMOUS
). Molecular cloning of these genes revealed that, except AP2
, they all represent MIKC-type MADS-box genes encoding transcription factors (MADS-domain proteins) (for a review see Theißen, 2001
). Thus the products of the ABC genes probably control the transcription of other genes (‘target genes’) whose products are directly or indirectly involved in the formation or function of floral organs.
MIKC-type proteins have a characteristic domain structure, including, from N- to C-terminus, a MADS (M-), intervening (I-), keratin-like (K-) and C-terminal (C-) domain (Münster et al., 1997
). In all kinds of MADS-domain proteins, the MADS-domain is by far the most highly conserved region; it is the major determinant of DNA binding, but it also performs functions in dimerization of MADS-domain proteins and in the binding of accessory factors. MADS-domain proteins bind to DNA sites with similarity to the consensus sequence 5′-CC(A/T)6
GG-3′, termed a ‘CArG-box’ (for ‘CC-A rich-GG’). The I-domain, located directly downstream of the MADS-domain, is relatively variable in length and not very strongly conserved. At least in some MIKC-type proteins, it constitutes an important determinant for the selective formation of DNA-binding dimers. The K-domain is characterized by a conserved, quite regular spacing of hydrophobic residues, which is proposed to allow for the formation of amphipathic helices. These are assumed to interact with amphipathic helices of other K-domain-containing proteins via the formation of coiled-coils to promote protein dimerization or multimeric complex formation (Kaufmann et al., 2005
). The most variable region is the C-domain at the C-terminal end of MIKC-type proteins. In some MIKC-type proteins it is involved in transcriptional activation, or in the formation of multimeric complexes (structural and phylogenetic aspects of MIKC-type proteins have been reviewed in detail by Kaufmann et al., 2005
Despite its heuristic value, the ABC model has two major shortcomings: mutant and transgenic studies revealed that the ABC genes are required, but not sufficient for the specification of floral organ identity. Moreover, the ABC model does not provide a molecular mechanism for the specification of organ identity by the interaction of floral homeotic genes. Development of the ‘ABCDE’ and the ‘floral quartet’ models allowed both of these shortcomings to be overcome. Based on the analysis of multiple mutants generated via ‘reverse genetics’, the ABC model was first extended to an ‘ABCD model’ by addition of class D genes that are closely related to class C genes and specify ovule identity (Angenent and Colombo, 1996
). Further consideration of class D genes is not required in the framework of this review.
Knowing about another class of closely related MIKC-type MADS-box genes – originally known as AGL2
-like genes (Theißen et al., 1996
), but later renamed into SEPALLATA
)-like genes (Pelaz et al., 2000
), is essential here, however. Arabidopsis has four different SEP
-like genes, termed SEP1
. While all single and the double mutants of SEP
revealed only very weak, if any, deviations from wild-type phenotype, in sep1 sep2 sep3
triple mutants the organs in all whorls of the flower develop into sepals, and flower development becomes indeterminate (Pelaz et al., 2000
); in sep1 sep2 sep3 sep4
quadruple mutants vegetative leaves rather than sepals develop in all whorls of indeterminate flowers (Ditta et al., 2004
genes are still expressed in loss-of-function mutants of class B and class C genes, and the initial expression patterns of B and C class genes are not altered in the sep1 sep2 sep3
triple mutant (Pelaz et al., 2000
). Therefore, the SEP
genes are considered as yet another class of floral organ identity genes, termed class E genes (Theißen, 2001
), required for the development of all categories of floral organs.
According to the ABCDE model class A + E genes are required to specify sepals, A + B + E petals, B + C + E stamens, C + E carpels and D + E ovules (Theißen, 2001
; Ditta et al., 2004
). But by which molecular mechanism do the different floral homeotic genes interact?
All MADS-domain proteins tested bind to DNA in a sequence-specific way as dimers. Explaining the interaction of floral homeotic genes by dimerization of their gene products soon failed, however, because the expected dimers were not observed in respective studies (Riechmann et al., 1996
; Fan et al., 1997
). For example, the class B proteins AP3 and PI bind to DNA only as obligate AP3–PI heterodimers, but DNA-binding dimers between AP3 or PI and AP1 (class A) or AG (class C) proteins were not observed (Riechmann et al., 1996
). An obvious way to explain the combinatorial interactions would be that class A and class B proteins (for specifying petals) or class B and class C proteins (for specifying stamens) bind separately to different cis
-regulatory elements in the promoters of the same target genes that are activated or repressed during petal or stamen formation, respectively. Alternatively, class A and class B (class B and class C) proteins could have distinct sets of target genes involved in petal (stamen) formation, so that the combinatorial interaction of the homeotic genes would be mechanistically realized only at the level of target genes, or even further downstream in the gene cascades involved.
Things turned out to be more interesting. An important clue was provided when Egea-Cortines et al.
(1999) reported that DEFICIENS (DEF), GLOBOSA (GLO) and SQUAMOSA (SQUA) from snapdragon (Antirrhinum majus
) – putative orthologues of AP3, PI and AP1, respectively – form multimeric complexes in electrophoretic mobility shift assays and yeast three-hybrid analyses. The authors hypothesized that the protein complex is actually a protein tetramer, composed of a DEF–GLO heterodimer and a SQUA–SQUA homodimer, in which the DEF–GLO and SQUA–SQUA dimers recognize different CArG-boxes (Egea-Cortines et al., 1999
When Pelaz et al.
(2000) reported that not only the ABC but also the SEP
genes are required for the formation of petals, stamens and carpels, the available data about the mechanisms of floral organ specification were pulled together in the ‘floral quartet model’ (Theißen, 2001
). It suggests that complexes of four floral homeotic proteins including SEP proteins control floral organ identity. According to the original model there is at least one unique quaternary complex for each type of floral organ (Theißen, 2001
). These quaternary protein complexes might function as transcription factors by binding to CArG-boxes in the promoters of target genes, which they either activate or repress as appropriate for the development of the identities of the different floral organs (Theißen, 2001
). There is evidence that in these complexes, the AP1 or SEP proteins provide the transcription-activation domain, while the other proteins might be important for organ-specificity of gene regulation (Honma and Goto, 2001
). The floral quartet model suggests that two protein dimers of each tetramer recognize two different CArG-boxes, which might be brought into close vicinity by bending the DNA between the CArG-boxes (Egea-Cortines et al., 1999
; Theißen, 2001
; Theißen and Saedler, 2001
), so the quartet (or tetramer) is actually a dimer of dimers.
The floral quartet model was soon corroborated by Honma and Goto (2001)
who demonstrated the formation of the complexes postulated for stamens and petals, namely AP3/PI/AG/SEP and AP3/PI/AP1 (or SEP), respectively, in yeast three-hybrid and four-hybrid assays. Moreover, it was shown that ectopic co-expression of class A/E + B genes AP1
in transgenic Arabidopsis plants leads to a reprogramming of supposed-to-be leaf primordia in a way that they develop into petaloid organs. AP1
can substitute each others which probably reflects their close evolutionary relationship (Becker and Theißen, 2003
) and hence partial redundancy (Honma and Goto, 2001
; Pelaz et al., 2001
). The co-expression of the class B + C + E genes AP3
converts vegetative leaves into stamenoid organs (Honma and Goto, 2001
). These data add not only support for the floral quartet model but also demonstrate that just a few proteins are both necessary and sufficient to superimpose petal and stamen identity upon a vegetative developmental programme.
Clearly, the empirical basis of the floral quartet model is still quite weak, especially since there is no direct evidence so far that multimeric complexes of floral homeotic proteins really exist, loop DNA, and regulate target gene expression in the nuclei of plant cells. Moreover, a number of important questions remain unanswered by the floral quartet model, for example, how target gene specificity is achieved by floral homeotic proteins (Melzer et al., 2006
). Limitations and possible experimental tests of the floral quartet model are discussed by Theißen and Melzer (2006)
Nevertheless, since the floral quartet model, in contrast to the ABCDE model, demonstrates how the combinatorial interaction of the floral homeotic genes works mechanistically, it has been widely accepted and became the standard model for work on floral homeotic genes (see, for example, Jack, 2001
; Winter et al., 2002a
; Ferrario et al., 2003
; Lee et al., 2003
; Ferrario et al., 2004
; Shchennikova et al., 2004
; Krizek and Fletcher, 2005
; Robles and Pelaz, 2005
). The floral quartet model may thus facilitate research on flower development and evolution in a similar way as the ABC model did 10 years before.
In the following, we outline the impact of the floral quartet model and the ABC model on understanding flower origin and diversification, respectively.