Annals of Botany 104: 809 –822, 2009
doi:10.1093/aob/mcp162, available online at www.aob.oxfordjournals.org
Establishment of zygomorphy on an ontogenic spiral and evolution of perianth
in the tribe Delphinieae (Ranunculaceae)
Florian Jabbour1,2,*, Louis P. Ronse De Craene3, Sophie Nadot1 and Catherine Damerval2
1
Université Paris-Sud, Laboratoire Ecologie, Systématique, Evolution, CNRS UMR 8079, AgroParisTech, Orsay, F-91405,
France, 2UMR de Génétique Végétale, INRA – Univ Paris-Sud – CNRS – AgroParisTech, Ferme du Moulon, 91190 Gif-surYvette, France and 3Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, UK
Received: 15 March 2009 Returned for revision: 29 April 2009 Accepted: 3 June 2009 Published electronically: 16 July 2009
† Background and Aims Ranunculaceae presents both ancestral and derived floral traits for eudicots, and as such
is of potential interest to understand key steps involved in the evolution of zygomorphy in eudicots. Zygomorphy
evolved once in Ranunculaceae, in the speciose and derived tribe Delphinieae. This tribe consists of two genera
(Aconitum and Delphinium s.l.) comprising more than one-quarter of the species of the family. In this paper, the
establishment of zygomorphy during development was investigated to cast light on the origin and evolution of
this morphological novelty.
† Methods The floral developmental sequence of six species of Ranunculaceae, three actinomorphic (Nigella
damascena, Aquilegia alpina and Clematis recta) and three zygomorphic (Aconitum napellus, Delphinium
staphisagria and D. grandiflorum), was compared. A developmental model was elaborated to break down the
successive acquisitions of floral organ identities on the ontogenic spiral (all the species studied except
Aquilegia have a spiral phyllotaxis), giving clues to understanding this complex morphogenesis from an evodevo point of view. In addition, the evolution of symmetry in Ranunculaceae was examined in conjunction
with other traits of flowers and with ecological factors.
† Key Results In the species studied, zygomorphy is established after organogenesis is completed, and is late,
compared with other zygomorphic eudicot species. Zygomorphy occurs in flowers characterized by a fixed
merism and a partially reduced and transformed corolla.
† Conclusions It is suggested that shifts in expression of genes controlling the merism, as well as floral symmetry
and organ identity, have played a critical role in the evolution of zygomorphy in Delphinieae, while the presence
of pollinators able to exploit the peculiar morphology of the flower has been a key factor for the maintenance and
diversification of this trait.
Key words: Delphinieae, development, evolution, evo-devo, nectar spurs, ontogenic spiral, Ranunculaceae,
zygomorphy.
IN T RO DU C T IO N
To understand the origin, establishment and evolution of
morphological novelties, it is crucial to study these new traits
from a developmental point of view. A morphological
novelty is considered a key innovation when the acquisition
of the new trait is correlated with unexpectedly high rates of
speciation in the clade housing the innovation (Sanderson and
Donoghue, 1994). In the field of plant biology, floral symmetry
is a well-known character for favouring evolutionary radiation
and rapid diversification (Sargent, 2004). This trait is generally
recorded for the perianth and androecium, the gynoecium often
being characterized by loss of organs (Endress, 1999). Two
principal types of symmetry are recognized among angiosperms: actinomorphy (radial symmetry or polysymmetry) and
zygomorphy (bilateral symmetry or monosymmetry). In an
actinomorphic flower, all organs of the same identity (sepal,
petal or stamen) in a whorl exhibit equal (or almost equal)
size and shape, and are inserted regularly on the receptacle.
An actinomorphic flower is defined by at least two identical
symmetry planes. By contrast, zygomorphy is defined by
* For correspondence. E-mail [email protected]
a supplementary identity superimposed on the basic organ identity, resulting in intra-whorl differentiation that generates a
single symmetry plane, most often dorsoventrally oriented.
Zygomorphy is a derived state that has arisen several times
independently in flowering plants (Endress, 1999; Cubas,
2004). Exceptions to actinomorphy and zygomorphy are relatively rare and occur in only a few taxa, such as some
Fumarioideae of Papaveraceae that have disymmetric flowers
(characterized by two unequal planes of symmetry, e.g.
Hypecoum and Dicentra sensu lato) or some Leguminosae,
Caprifoliaceae and Cannaceae that possess asymmetric
flowers (Dahlgren et al., 1985; Endress, 1999; Tucker, 2003).
Whereas in basal angiosperms transitions towards zygomorphy are scarce (e.g. Piperales), such transitions are much more
frequent in the eudicots. Among basal eudicots, the order
Ranunculales is sister to all other eudicots, and possesses features that are found both in basal angiosperms and in the morederived eudicots. Three independent acquisitions of zygomorphy have been recorded within the Ranunculales, each in a
different family (Papaveraceae, Menispermaceae and
Ranunculaceae; Damerval and Nadot, 2007). Here, the focus
is on Ranunculaceae (62 genera, 2525 species), a family that
presents both ancestral and derived floral traits for eudicots,
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
and as such is of potential interest to understand key steps
involved in the evolution of zygomorphy in eudicots. In
Ranunculaceae, at least some floral organs are initiated on a
spiral. Spiral phyllotaxis is an ancestral state and is traditionally associated with actinomorphy. Interestingly, in
Ranunculaceae, zygomorphy takes place only in flowers with
spiral phyllotaxis. The transition to zygomorphy occurred
once in the family, in the ancestor of three sister genera,
Aconitum, Delphinium and Consolida, which form the tribe
Delphinieae. The genera Aconitum and Delphinium s.l.
(Delphinium þ Consolida) comprise approx. 300 and 365
species, respectively, which in total accounts for approx.
26 % of all Ranunculaceae species [Tamura, 1993; also electronic databases: eFloras (www.efloras.org), Angiosperm
Phylogeny Website (Stevens, 2001) and Delta (Watson and
Dallwitz, 1992 onwards)]. In all species of Delphinieae, zygomorphy is apparent primarily in the calyx and corolla, where
the dorso-lateral/ventral petals are reduced and the two
adaxial ones develop into nectar spurs.
In order to understand the developmental and evolutionary
bases of zygomorphy in Delphinieae, floral organogenesis
and organ development is analysed and compared in six
species, three with actinomorphic flowers (Nigella damascena,
Aquilegia alpina and Clematis recta) and three with zygomorphic flowers (Aconitum napellus, Delphinium staphisagria
and D. grandiflorum). Full or partial descriptions have already
been published for Aquilegia (Tucker and Hodges, 2005) and
Aconitum (development of the petals: Kosuge, 1994; Kosuge
and Tamura, 1988). This study concentrates on the timing of
events leading to the establishment of zygomorphy and the formation of spurs. Moreover, the changes in organ identities
during organogenesis are analysed in order to determine
(a) the factors that can discriminate between two different
and consecutive organs on the spiral and (b) the developmental
origins of different structures in the corolla. The floral development of Delphinieae is compared with that of two zygomorphic species belonging to different eudicot lineages (the
derived core eudicot Antirrhinum majus of Plantaginaceae
and the basal eudicot Synaphea spinulosa of Proteaceae), in
order to identify potential specificities of Delphinieae. In
addition, the architectural and ecological contexts associated
with the emergence of zygomorphy in Ranunculaceae are
examined by looking at the evolution of floral and inflorescence architectural traits, the type of pollination and the
sexual system.
TA B L E 1. Species of Ranunculaceae and origin of accessions
used for developmental analyses
Species
Aconitum napellus L.
Aquilegia alpina L.
Clematis recta L.
Delphinium grandiflorum L.
Delphinium staphisagria L.
Nigella damascena L.
Origin
Horticultural
Horticultural
Horticultural
Horticultural
Accession 02.58, Natural History Museum,
Paris
Accession 04.98, Natural History Museum,
Paris
this genus because of the particular shape of the androecium.
Except C. recta that exhibits staminodes between calyx and
androecium, the five other species have petals more or less
developed. Among the zygomorphic taxa, the genus
Delphinium presents flowers with two dorsal spurred petals
bearing nectaries inside the spurs. They also have two dorsolateral hairy flat petals without nectaries and four reduced
ventral petals. Spurred petals are short in Delphinium staphisagria, but long in D. grandiflorum, and both are wrapped in a
single spurred sepal. Aconitum napellus presents a hoodshaped sepal enclosing two spurred nectariferous petals, and
six reduced petals without nectaries. In addition to the dorsal
sepal, the pentamerous calyx of Delphinieae consists of two
dorso-lateral and two ventral sepals.
Two eudicot species were chosen in order to represent the
establishment of zygomorphy in taxa presenting various
degrees of relatedness with the Ranunculaceae and their developmental sequence was reconstructed from literature. Synaphea
spinulosa (Proteaceae; Douglas, 1997) was chosen because
Proteales belong to the basal eudicot grade like Ranunculales,
whereas Antirrhinum majus (Plantaginaceae; Vincent and
Coen, 2004) belongs to a derived clade of the core eudicots.
Plants were grown in a greenhouse at UMR de Génétique
Végétale, Gif-sur-Yvette, France. Floral buds were sampled
in order to obtain an exhaustive representation of the developmental sequence. They were immediately fixed in FAA
(85 mL 55 % ethanol/5 mL glacial acetic acid/10 mL formaldehyde) and then stored in 70 % ethanol. Transverse sections
of Ac. napellus were observed with a binocular microscope
Zeiss SV11.
Microscopy and calibration of development sequence over time
M AT E R IA L S A ND M E T HO DS
Species sampled and material collection
The taxa investigated are listed in Table 1. The six species
were chosen for their morphological specificities (Fig. 1).
All six species have petaloid sepals. The three actinomorphic
species have a polysymmetric perianth but, in addition to
this, Nigella damascena presents a series of bulged petals
forming hairs and bearing nectaries, Aquilegia alpina has a
whorled phyllotaxis and as many spurred petals as indicated
by merism, and Clematis recta has no corolla. It should be
noted that C. recta will be considered actinomorphic even
though Endress (1987) described symmetry as bilateral in
Buds were dissected with a Wild MZ8 stereomicroscope
(Leica, Wetzlar, Germany), dehydrated in an ethanol-acetone
series, and dried with a K850 critical point dryer (Emitech,
Ashford, Kent, UK). Dried floral structures were mounted on
aluminium stubs with colloidal graphite and coated with platinum using an Emitech K575X sputter coater (Emitech) and
observed with a Supra 55VP scanning electron microscope
(LEO Electron Microscopy, Cambridge, UK). Pickled reference material and platinum coated material are kept at UMR
de Génétique Végétale.
The successive developmental stages were represented
along an axis of relative time representing the development
of the flower, from the initiation of the first sepal primordium
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
A1
B1
C1
D1
E1
811
F1
F2
B2
A2
C2
E2
D2
F I G . 1. Photographs of the species studied. Actinomorphic species: Clematis recta (A1, A2), Nigella damascena (B1, B2) and Aquilegia alpina (C1, C2); zygomorphic species: Delphinium grandiflorum (D1, D2), D. staphisagria (E1, E2) and Aconitum napellus (F1, F2). Arrows indicate: the bulged petals in
N. damascena (B); the spurred petals in Aq. alpina (C); the location of the spurred sepal and of the pair of spurred petals in D. staphisagria (E1, E2, respectively).
Scale bars: (A, B) ¼ 0.5 cm; (C–F) ¼ 1 cm.
TA B L E 2. Species of Delphinieae and Nigella and available data about their pollination compiled from the literature
Species
Spur
length
(mm)
D. nuttallianum
8– 23
D. barbeyi
Inventoried pollinators/visitors
Diptera
Solitary
bees
Hymenoptera
Apodiformes
Queen bumblebees
Hummingbirds
10–18
Bumblebees
Hummingbirds
D. decorum
D. nudicaule
D. leroyi
Ac. columbianum
19.3– 28.2
26–33.4
30–40
approx. 11
Bumblebees
Ac. lycoctonum
Ac. gymnandrum
Ac. septentrionale
16–20
approx. 18
approx. 24
Bombus gerstaeckeri
Bumblebees
Specialist Bombus consobrinus
Nigella arvensis
No spur
Lepidoptera
References
Hawkmoths
Bosch and Waser, 1999; Williams et al.,
2001; Flora of North America (eFloras:
www.efloras.org)
Williams et al., 2001; Flora of North
America (eFloras)
Guerrant, 1982
Guerrant, 1982
Johnson, 2001; Flora Zambesiaca (eFloras)
Bosch and Waser, 1999; Flora of North
America (eFloras)
Utelli et al., 2000
Zhang et al., 2006; Flora of China (eFloras)
Thostesen and Olesen, 1996; Flora of
China (eFloras)
Weber, 1995
Hummingbirds
Queen and worker bumblebees
Apis
mellifera
Pyrobombus lapidarius,
Polistes dominulus, Eumenes
pedonculatus
on the floral apex to the bud just before blooming. Time was
calibrated according to the size of the floral receptacle. This
organ grows continuously and homogeneously, thus giving a
reliable image of the pace of development (see Douglas,
1997). The relative time was calculated as the proportion
between the diameter of the receptacle of the bud and that of
the adult open flower. The diameter of the floral receptacle
for buds and adult flower was measured for several individuals
and plants (three to seven from different plants).
Reconstruction of character evolution
Morphological data ( phyllotaxis, merism, number of nectar
spurs, sexual system and type of inflorescence) were compiled
from Tamura (1993) and electronic databases: eFloras (www
.efloras.org), Angiosperm Phylogeny Website (Stevens, 2001
onwards) and Delta (Watson and Dallwitz, 1992 onwards).
Pollination data were compiled from the literature (Table 2).
A matrix of 31 taxa including all characters was built using
Mesquite 2.01 (Maddison, 2000). Character states were
plotted on the most recent phylogenetic tree of Ranunculaceae, comprising 31 genera among the 62 identified (Hoot,
2008). Ancestral states were not resolved due to the lack of resolution at the deepest nodes of the tree.
R E S ULT S
Comparative developmental analysis was done in six species of
Ranunculaceae, in order to describe precisely the organogenesis
and further differentiation of floral organs preceding the establishment of zygomorphy. Three species of Delphinieae (one
species of Aconitum and two of Delphinium differing by the
length of the spur), one species of Nigella, that is considered
the sister genus of Delphinieae or at least being a part of their
sister clade, one species of Aquilegia (Aq. alpina) that shares
architectural traits with Nigella but is phylogenetically distant
from Delphinieae, and one species of Clematis where a corolla
is lacking (C. recta) were selected.
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Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
Timing of events during floral development
The developmental sequence of the six species studied is
presented schematically in Fig. 2, highlighting the various
times when zygomorphy is established and spurs are formed
in the Delphinieae species. Developmental stages are shown
for the three zygomorphic species (Figs 3, 4 and 5, and
Fig. S1 in Supplementary data, available online).
The initiation of all organs is most generally completed during
the first quarter of development. In all species studied floral organ
primordia initiate in a helical fashion (Figs 3 and 6, and Fig. S2 in
Supplementary data), with the exception of petals, stamens and
carpels in Aq. alpina which initiate in a whorled fashion (Fig.
S2 in Supplementary data), and sepals in C. recta that appear as
two opposite decussate primordia (data not shown).
In Delphinieae and Nigella, the perianth is composed of five
petaloid sepals and eight more or less reduced petals. Sepal,
petal and stamen initiation consists of four, seven and n 2 1 plastochrons, respectively, n being the number of stamens. The sepals
and petals initiate on respectively two and three revolutions of the
spiral, meaning that phyllotaxis changes from 2/5 to 3/8 from
calyx to corolla. The time devoted to the initiation of the three
types of organs is equivalent (Fig. 2). This means that four plastochrons (separating the initiation of five sepals) must last as
long as seven plastochrons (separating the initiation of eight
petals). Since calyx and corolla appeared whorled in adult
flowers, the plastochron between the last sepal and the first petal
must be longer. It can therefore be concluded that petals initiate
more rapidly than sepals and that stamens initiate even more
rapidly than petals (if n . 8).
Aquilegia alpina bears five petals that initiate at the same
speed as sepals. However, stamens initiate more rapidly, as
in the other species studied. This developmental sequence is
identical to that described by Tucker and Hodges (2005) for
Aq. olympica. As noticed by Kramer and Hall (2005),
staminodes are generally found between stamens and carpels.
After initiation, petals in Delphinieae, N. damascena and
Aq. alpina appear to be delayed in development compared
with the other organs. In Aq. alpina and N. damascena, they
acquire their adult form at around the first quarter of the developmental sequence, after organogenesis is completed. In zygomorphic species, the protuberances preceding the spurs appear
on the limb of the petals after all organs have been initiated.
The dorsal sepal becomes hooded (Ac. napellus) and spurred
(D. grandiflorum) a quarter time of development after the
emergence of spurs in the two dorsal petals. Kosuge and
Tamura (1988) noticed that zygomorphy is established when
the bud is approx. 2 mm, which is consistent with the
present results. Spurs in the dorsal petals and in the dorsal
sepal of D. staphisagria appear later than in D. grandiflorum.
Zygomorphy plays its cards close to its chest
Zygomorphy in the three Delphinieae species is established
late in development, compared with the situation in the two
outgroup species Synaphea spinulosa and Antirrhinum
majus, where zygomorphy is established during initiation of
the calyx (Douglas, 1997; Vincent and Coen, 2004). In the
tribe Delphinieae, the floral apex remains a circular and
Clematis recta
Aquilegia alpina
Nigella damascena
Aconitum napellus
Delphinium staphisagria
Delphinium grandiflorum
Synaphea spinulosa
Antirrhinum majus
0
First sepal
initiation
Initiation of sepals
Initiation of petals
Initiation of stamens
Initiation of carpels
0·25
0·5
0·75
Relative floral base width
1
Open
flower
Acquisition of the definitive form of petals (spurred in Aq. alpina, bulged in N. damascena)
Establishment of zygomorphy
Establishment of zygomorphy due to the formation of two nectar spurs
Acquisition of a hooded or a spurred shape in the dorsal sepal
F I G . 2. Timing of events during floral development in the six species of Ranunculaceae studied and in two eudicot species [Synaphea spinulosa (Proteaceae) and
Antirrhinum majus (Plantaginaceae)]. The relative time axis indicates the percentage of development already accomplished (calculated as floral base width of the
bud/floral base width of the adult flower). Striped rectangle in S. spinulosa refers to the initiation of tepals.
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
A
B
C
D
E
F
G
H
I
J
K
L
813
F I G . 3. SEM micrographs of the developmental sequence of Aconitum napellus. (A) Young inflorescence; (B) floral primordium showing the first three sepals
(after removal of the bract and of the two prophylls); (C, D) floral primordia showing the spiral initiation of sepals (numbers indicate the order of initiation of
sepals); (E) floral primordium showing the second and fifth petals (seventh and tenth perianth members, P7 and P10) initiating in front of the dorsal sepal, and a
very young stamen primordium (St); (F –H) floral primordia showing spiral initiation of stamens; (I) basal view of a floral bud showing reduced petals (Rp); (J)
side view of a floral bud showing the two spurred petals of different size (P7 and P10); (K) apical view of a floral bud showing three carpels in central position
surrounded by stamens; (L) side view of a floral bud showing a spurred petal and stamens. Scale bars: (A– K) ¼ 100 mm; (L) ¼ 1 mm.
814
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
A
B
F
K
C
G
L
D
H
M
N
E
I
J
O
P
F I G . 4. Transverse sections of floral buds of Aconitum napellus at 20 % (A) to 85 % (J) of development. Abbreviations: sk, stalk; lb, labium; sp, spur. (K) Young inflorescence; (L) inflorescence with petaloidy of sepals initiating; (M) side view of an adult flower; (N) frontal view of an adult flower; (O) adult flower with the dorsal sepal
removed; (P) side view of an adult flower with all sepals removed. Scale bars: (A, B) ¼ 0.5 mm; (C–E, G, K) ¼ 1 mm; (F) ¼ 2 mm, (H–J) ¼ 5 mm; (M) ¼ 1 cm.
regular mound until the end of androecium initiation.
Subsequently, dorsoventral zygomorphy is first generated by
reduced growth of petals in the abaxial region compared
with the dorsal petals, with the reduction of six petals (one
facing each dorso-lateral sepal and a pair facing each ventral
sepal) in the genus Aconitum and four (one pair facing each
ventral sepal) petals in the genus Delphinium (Figs 2 and 5,
and Fig. S1 in Supplementary data). The two dorsalmost
petals further develop into spurs.
Interestingly, zygomorphy becomes conspicuous from the
outside of the flower still later, when the stalks of the spurred
petals begin to grow (Fig. 4), and the dorsal sepal differentiates
into a helmet in Aconitum (Figs 3 and 4) and into a single spur
in Delphinium (Fig. 5, and Fig. S1 in Supplementary data). It
must be noticed that the elongation of the dorsal sepal does not
take place simultaneously with that of nectar spurs. Indeed, a
phase of latency with a specific duration for each species precedes the formation of the spur or hood in the dorsal sepal
(Fig. 2). Spurred petals reach their final length after anthesis.
Historical associations among floral and ecological characters
From the comparison of the evolution of symmetry and other
floral traits, it is shown that, in Ranunculaceae, zygomorphy
evolved in a relatively fixed ground-plan (at least with a stable
number of sepals or petals; Fig. 7) associated with pentamery
of the calyx and racemose inflorescences. The three zygomorphic
genera have polyandrous, bisexual flowers with a spiral phyllotaxis and a perianth differentiated between calyx and corolla.
According to the literature (see references in Table 2),
short-spurred flowers of Delphinieae are pollinated by bees
and bumblebees, those with medium-sized spurs by hummingbirds and those with long spurs by hawkmoths (Table 2). In
most cases, effective pollinators are not specialized visitors.
In the genus Nigella, visitors/pollinators were only identified
for the species N. arvensis and consist of Apis mellifera and
representatives of three genera of Hymenoptera: Pyrobombus,
Polistes and Eumenes (Weber, 1995). Delphinieae are thus
visited by a broader spectrum of animals (hawkmoths and hummingbirds in addition to Diptera and Hymenoptera).
D IS C US S IO N
Partial descriptions of floral organogenesis and organ
development in Ranunculaceae have already been published
(Payer, 1857; Mair, 1977; Kosuge and Tamura, 1988; Kosuge,
1994; Tucker, 1997; Tucker and Hodges, 2005; Erbar et al.,
1999; Ren et al., 2004; Song et al., 2007). The organogenesis
of the flower was not dealt with in a chronologically detailed
way in these studies, and the present work is the first one to
tackle this issue from the floral symmetry point of view. In
addition, the present study provides data on new species and
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
A
B
C
D
E
F
G
H
I
815
F I G . 5. SEM micrographs of the developmental sequence of Delphinium grandiflorum. (A) floral primordium showing the spiral initiation of sepals (numbers
from 1 to 5 indicate the order of initiation of sepals; the first sepal is removed); (B) floral primordium showing the first and fourth petal primordia (sixth and ninth
perianth members, P6 and P9) initiating in front of the dorsal sepal; (C, D) floral primordia showing spiral initiation of stamens; (E) initiation of three carpels in
the centre of the floral primordium; (F) side view of a floral bud showing a reduced petal (Rp) and what will become a flat petal (Fp) and a spurred petal (Sp) –
dorsoventral asymmetry is due to a unidirectional growth of petals indicated by the size of the black asterisks (the double white arrow gives the direction of the
median symmetry plane); (G, H) basal view of a floral bud showing the pair of flat petals, the pair of spurred petals and a reduced petal (in H). (I) side view of a
floral bud showing reduced petals and the stalks of stamens (St). Scale bars: (A– G, I) ¼ 100 mm; (H) ¼ 200 mm.
full developmental sequences which help to understand the
establishment of zygomorphy in a derived tribe, and the
distribution of different organ identities on an ontogenic spiral.
Acquisition of floral organ identities on an ontogenic spiral in
Nigella and Delphinieae
All the species studied have a centripetal organogenesis
(Schöffel, 1932), but they differ in the way organs are initiated,
on a full ontogenic spiral or not. Even though Aquilegia
species have a spirally arranged calyx, the corolla is whorled
and the stamens are initiated on orthostichies, a characteristic
of whorled phyllotaxis (Endress and Doyle, 2007). Petals are
the first organs initiated at the base of orthostichies. The four
sepals of Clematis species are initiated in pairs and not on a
spiral, as is the case for the androecium. Outer stamens arise
in whorls, but initiation becomes spiral higher on the apex
(Ronse De Craene and Smets, 1996). In Nigella and
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Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
N. damascena
Ac. napellus
D. grandiflorum
2
10
7
4
5
13
12
8
9
3
11
6
1
F I G . 6. Floral diagrams of Nigella damascena, Aconitum napellus and Delphinium grandiflorum. Black dot, the axis of the inflorescence; black triangles, bracts
(the two lateral ones in zygomorphic species are prophylls); grey crescents, sepals (the dorsalmost one in Delphinieae is deformed into a hood or a spur); hairy
crescents, bulged petals bearing nectaries in N. damascena; small blank crescents, reduced petals; bigger blank crescent, flat petals in D. grandiflorum; blank
ellipses, stamens. The gynoecium is at the centre of the flower diagram. In D. grandiflorum, numbers refer to the order of sepal and petal initiation.
Delphinieae, all organs are initiated on a same ontogenic
spiral. In most species studied, the various organs share
characteristics (like colour, cell type, shape, initiation timing,
appendages, etc.) during development resulting in blurred
identities, which is all the more problematic when all organs
are initiated on the same ontogenic spiral. In this context, comparative analysis of the developmental process in the various
species may help define criteria for identifying boundaries
between spirally arranged organs.
In the early stages of development of the species bearing
petals, petal primordia are different from sepal primordia in
shape and size. Moreover, the cell type may also help distinguish between both (Kramer et al., 2007). In contrast,
petal primordia are indistinguishable from stamen primordia,
exhibiting similar shape and size (Figs 3 and 5, and Fig. S1
in Supplementary data; Payer, 1857; Tepfer, 1953; Hiepko,
1965; Sattler, 1973; Kosuge and Tamura, 1988, 1989;
Kosuge, 1994). This similarity was also shown at the anatomical level (Payer, 1857; Smith, 1928; Hiepko, 1965; Tamura,
1965, 1984; Erbar et al., 1999). The link of petals with staminodes (andropetals) in Ranunculaceae was hypothesized by
Ronse De Craene and Smets (1994, 1995) and Erbar et al.
(1999), based on morphological and developmental observations. Petals in Ranunculaceae were considered as the outermost whorl of the androecium, and interpreted in a
phylogenetic sense as heterotopic staminodes (Ronse De
Craene and Smets, 2001; see Ronse De Craene, 2003). An
alternative hypothesis has been presented by a recent developmental genetic study of Rasmussen et al. (2009). The authors
have shown that petals in the whole order Ranunculales must
not be seen as homoplastic derived staminodes but rather as
the result of a genetic developmental programme, common
to Berberidaceae and Ranunculaceae. Gradients of gene
expression may lead to the morphological grades (Kunst
et al., 1989) between stamens and petals that were interpreted
in the past as reproducing the evolutionary history between
stamens and petals.
In Nigella, Aquilegia and Delphinieae, the growth pace and
differentiation of petal and stamen primordia differ following
organogenesis. Stamen primordia soon differentiate into filament and anther, while the growth of petal primordia is
delayed. The retarded development of petals is a common
feature of other plant families (Payer, 1857; Sattler, 1973)
and has been explained as the arrested condition of original
stamens (e.g. de Candolle, 1817; Troll, 1928; Arber, 1937).
Petals develop once all organs have been initiated. The late
development of three-dimensional structures such as the
spurred/bulged petals confers a specific and supplementary
identity to petals of Aquilegia, Nigella and the Delphinieae
among Ranunculaceae, which appears to be linked to the
presence of nectaries on petals.
Thus, the present study in Ranunculaceae species helps us
define morphological and developmental criteria to distinguish
the identity of initiating organs juxtaposed on an ontogenic
spiral: (a) the relative position of organs on the spiral (depending on this position the organ which initiates after a sepal is
either a sepal or a petal); (b) the longer plastochron between
two organs of two different identities compared with two
similar organs; (c) the pace of organogenesis ( petals and
stamens initiates more rapidly than sepals); and (d ) the delay
in petal development compared with other floral organs.
A developmental model is proposed that synthesizes these
changes in floral identities along the ontogenic spiral, and
hypothesizes the acquisition of supplementary specific identities for petals that account for the emergence of zygomorphy
in tribe Delphinieae (Fig. 8). The sister taxon to Delphinieae
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
817
Glaucidium
Hydrastis
Xanthorhiza
Coptis
Thalictrum
Enemion
Semiaquilegia
Aquilegia
Trollius
Adonis
Nigella
Aconitum
Consolida
Delphinium
Helleborus
Anemonopsis
Actaea
Cimicifuga
Eranthis
Caltha
Anemone
Clematis
Ranunculus 1
Hamadryas
Halerpestes
Trautvetteria
Ceratocephala
Myosurus
Ranunculus 2
Krapfia
Laccopetalum
F I G . 7. Evolution of perianth merism represented on the phylogenetic tree of Ranunculaceae in Hoot (2008). Dark blue, dimerous; light blue, tetramerous;
yellow, trimerous; green, pentamerous; black, variable. The tribe Delphinieae is highlighted in pink.
818
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
Petal identity = (bulged
/spurred, nectariferous,
hairy)
Petal identity =
(bulged/spurred,
nectariferous, not hairy)
Delphinium
Dorsal sepal identity
=
(spurred).
Petal identity =
(flat and hairy)
A
Petal identity =
(retarded growth
compared to other
floral organs)
B
Development of petals in
association with the expression of
C
a morphogenetic factor. This
factor is expressed all over the
corolla or following a dorso-ventral
gradient in Delphinieae.
Nigella
Petal identity = (bulged
/spurred, nectariferous,
not hairy)
Aconitum
D
: developing petals.
: still reduced petals.
F I G . 8. Schematic sequence of events during floral development of Nigella and Delphinieae. A series of developing meristems is shown. The concentric discs
represent the orthogonal projection of the ontogenic spiral onto a plane. This is not a growth model so the size of meristems does not vary. Nigella is represented
with eight petals to make the comparison with Delphinieae easier (the number of petals in Nigella may range between zero and ten, although eight is most
common). Green crescents, sepals; blue crescents, petaloid sepals; light violet crescents, dorso-lateral sepals; grey crescents, ventral sepals; light blue disc,
androecium; brown disc, gynoecium.
was considered to be Nigella (Hoot, 1991, 1995), even though
the clade formed by Nigella, Actaea and Cimicifuga has
also been suggested by some authors (Johansson and
Jansen, 1993). After organogenesis and when ‘sepal’, ‘petal’,
‘stamen’ and ‘carpel’ identities are established (Fig. 8A),
Nigella flowers retain an actinomorphic corolla, while
flowers of Delphinieae acquire a dorsoventral asymmetry as
a result of the unidirectional growth of petals (see Figs 5F
and 8B): the abaxial petals are reduced and the adaxial ones
develop into petaloid structures. This bilateral symmetry is
not perfect, however, due to the spiral initiation of petal primordia. This phenotype could be determined by a morphogen
present in a dorsoventral concentration gradient (see Fig. 5f )
acting either as a growth repressor, which is more abundant
in the abaxial region, or conversely acting as an activator,
which is more abundant in the dorsal region. The asymmetry
pattern thus differs from the pattern described in An. majus,
where development is retarded in the dorsal petals and
stamen due to asymmetric early expression of the
CYCLOIDEA (CYC) growth repressor gene (Luo et al.,
1996). The effect of CYC in Antirrhinum appears to be
organ and stage specific: retarded/suppressed growth of the
dorsal stamen, retarded dorsal petal growth during early development but enlargement of dorsal petals compared with dorsolateral/ventral ones during late development. The expression of
CYC-like genes has been recorded in ventral petals of a few
core eudicots (Howarth and Donoghue, 2006; Broholm
et al., 2008) suggesting that these genes may also play a role
in ventral identity in some species.
As development proceeds, all petals of Nigella become
bulged and nectariferous, and form hairs on two flat appendages (Fig. 9B). These two features are observed in different
petals in Delphinium. The petals in dorso-lateral position
become hairy but harbour a flat shape. The two dorsalmost
petals of Delphinium, and also of Aconitum take a structure
similar to that of bulged petals of Nigella, the petal claws probably being homologous, as well as the bulge and the growing
spur of dorsal petals (Fig. 9). Hairs are absent from all petals in
Aconitum (Fig. 8C). If the Nigella morph is indeed ancestral,
the corolla of Delphinieae would result from the reduction of
four (Delphinium) or six (Aconitum) petals in conjunction
with the differentiation of partly new petal identities. The distinct identities ‘spurred and nectariferous’ (in Aconitum and
Delphinium) and ‘flat, hairy and non-nectariferous’ (in
Delphinium) could be considered as a split of a composite
identity (‘bulged, hairy and nectariferous’) specific to the
petals of Nigella.
The development of the petal spurs in Delphinium and
Aconitum precedes the acquisition of a spur in the dorsal
sepal (Fig. 8D). Indeed, the growth of this sepal may depend
on the same morphogenetic factor that induces the formation
of nectar spurs, but expressed later. At the end of organ development, the calyx of all species studied becomes petaloid in
shape and pigmentation (Fig. 8D).
In model core eudicot species, petal identity is controlled by
the interplay between different MADS-box genes, more specifically the so-called B-class genes (Coen and Meyerowitz, 1991).
They consist of two main lineages, APETALA3/DEFICIENS
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
A
B
C
D
E
F
819
F I G . 9. SEM micrographs of the changes in the morphology of the petals in Nigella damascena (A– C) and Aconitum napellus (D–F) through development.
Abbreviations: Sk, Stalk; Lp, lip; Bg, bulges. Scale bars: (A, B, D–F) ¼ 100 mm; (C) ¼ 200 mm.
(AP3/DEF) and PISTILLATA/GLOBOSA (PI/GLO) that have
originated from a duplication predating the emergence of angiosperms (Kramer et al., 1998). Both lineages have been extensively
studied, especially in the Ranunculaceae by the group of
E. Kramer (Kramer et al., 1998, 2003, 2007; Kramer and Irish,
1999, 2000; Rasmussen et al., 2009). Two duplications took
place in the AP3 lineage before the diversification of the
Ranunculales, giving rise to three paralogous lineages, while
the PI lineage underwent several more recent duplications
(Kramer et al., 2003; Rasmussen et al., 2009). Expression of
AP3-I and AP3-II in sepals, together with PI, evolved early in
Ranunculales but was lost many times, and lines of evidence
for a role of B-gene expression in sepal petaloidy are faint.
Expression of AP3-III lineage genes was found closely correlated
with the presence of petals in Ranunculales, and a petal-specific
expression of AP3-III genes appears in both Berberidaceae and
Ranunculaceae. Recent expression studies suggest that AP3-III
orthologues are more expressed in dorsal petals than in dorsolateral ones in Delphinium exaltatum (Rasmussen et al., 2009).
Whether this asymmetric expression could correspond to our postulated morphogen gradient or could just be a response to it
remains open. Moreover the genetic and molecular background
for three-dimensional appendages of petals such as spurs,
have still not been deciphered, although the redeployment of
expression of KNOX genes controlling petal tube formation has
been suggested in Antirrhineae (Golz et al., 2002).
Specificities of floral symmetry in Ranunculaceae
Floral symmetry in Ranunculaceae (whether actinomorphic
or zygomorphic) is actually imperfect due to spiral initiation
(Endress, 1999) and a homogeneous growth resulting in
unequal size among organs sharing a same identity
[in Aconitum (Fig. 3) and Delphinium (Fig. 5, and Fig. S1 in
Supplementary data) the growth of one spurred petal is
delayed compared with the other]. This situation is specific
to Ranunculaceae.
Zygomorphy follows the same architectural plan in the three
genera (of which only two were studied here) of Delphinieae.
In the corolla, it is linked to asymetric growth and the development of two dorsal spurs. Interestingly, flowers with
paired spurs are extremely rare among angiosperms. As far
as is known, the genus Diascia (Scrophulariaceae) is the
only other example of a flower with paired spurs. In the
calyx, zygomorphy is linked to a single spur whose development may be promoted by the genetic programme already
responsible for the dorso-ventral differentiation in the
corolla. Different spur lengths have been recorded in the
820
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
genus Delphinium. The difference between D. grandiflorum
and D. staphisagria can be interpreted as a heterochrony
since the spurs emerge later in D. staphisagria than in
D. grandiflorum but grow at the same rate in both species
according to the observations made (Fig. 2). Another case of
heterochrony was recorded in D. nudicaule, where the
corolla resembles the buds of many other Delphinium
species (Guerrant, 1982).
In Delphinieae, the floral apex is actinomorphic until the
end of organogenesis, and zygomorphy appears after all
organs have been initiated (also observed by Tucker, 1997),
which is quite late compared with eudicot zygomorphic
species (see the developmental sequence of the other basal
eudicot species S. spinulosa and of the core eudicot An.
majus in Fig. 2). For example, in the model species
An. majus (Plantaginaceae), zygomorphy is established at the
15th plastochron among the 58 identified by Vincent and
Coen (2004), i.e. between 0 % and 9 % of the developmental
sequence, with the differentiation of dorsal, lateral and
ventral sepals. The study of Feng et al. (2006) on Lotus japonicus (Fabaceae) shows that zygomorphy is established at the
very beginning of floral development when the floral organs
initiate in a unidirectional order (Dong et al., 2005). It has
been suggested that late establishment of zygomorphy during
development characterizes species occurring in groups with
predominantly actinomorphic flowers (Endress, 1999), which
is the case in Ranunculaceae. Similarly, in Iberis amara, a
species that belongs to a family with otherwise actinomorphic
(or disymmetric) flower species (Brassicaceae, Rosidae), petals
begin to grow in a heterogeneous way only after the onset of
stamen initiation (Busch and Zachgo, 2007).
Accounting for the evolutionary success of Delphinieae:
evolutionary forces driving floral symmetry in Ranunculaceae
The ancestral states for the flowers in Ranunculaceae
include spiral phyllotaxis (Hirmer, 1931; Schöffel, 1932) and
an open floral ground-plan (with a variable number of
organs of each type), two conditions traditionally only associated with actinomorphy (Endress, 1987). Fixed merism
evolved at least nine times in the family (Fig. 7) but only
once, in conjunction with partial reduction of the corolla, in
the ancestor of Delphinieae. In this case, the spurred petals
bearing nectaries can develop and grow in the bud with less
spatial constraints, generating flower zygomorphy. Even if
the sister taxa of Delphinieae may present flowers with a
number of sepals different from five and a number of petals
different from eight, it is assumed that a fixed merism preceded
the evolution towards zygomorphy and is a prerequisite to it
[consistent with the hypothesis of Endress (2001); see
Jabbour et al. (2008)].
Zygomorphy evolved in Delphinieae in conjunction with
nectar spurs. Both of these traits are considered key innovations in angiosperms (Hodges, 1997; Hodges and Arnold,
1995; Sargent, 2004). Disentangling the respective evolutionary role of zygomorphy and nectar spurs would require
studies assessing which is the initial attractor for pollinators:
is it the volatile chemical components of nectar (chemical
attraction) or the zygomorphy of the flower (visual attraction)?
Data are lacking in this field of study and it is consequently
impossible to know which character played the primary role
in species diversification. Nectar spurs and zygomorphy will
therefore be considered as a single morphological novelty.
The question is whether this morphological novelty represents
a key innovation in this tribe. No case of reversal to actinomorphy is recorded among the 665 species that make up the
Delphinieae, suggesting that ‘zygomorphy þ nectar spurs’
can be considered a key innovation according to the qualitative
definition proposed by Endress (2001). Nevertheless, accelerated speciation can simply be due to stochastic processes,
and the hypotheses of key innovation must then be statistically
tested for changes in speciation rates (Raup et al., 1973;
Slowinski and Guyer, 1989, 1993). Using several statistical
tests, Hodges and Arnold (1995) showed that the answer to
the question is dependent on the sister group of Delphinieae,
emphasizing the need for a better resolved phylogenetic tree
of Ranunculaceae to definitely clarify this point.
It is tempting to suggest that the evolution of zygomorphy is
dependent on the animals able to pollinate these new-shaped
flowers. Some regular flowers of Ranunculaceae may have
evolved repeatedly to shapes with deviations from actinomorphy, but ecological conditions may have been unfavourable for
the maintenance of zygomorphy in most cases except in
Delphinieae. Accordingly, spur length appears to be a critical
factor in animal – plant coevolution. According to the ‘pollinator shift’ hypothesis of Ennos (2008), spur length remains
stable or increases through coevolution with pollinators. It
would be very interesting to test this assertion using a detailed
phylogeny of Delphinieae, to see if species with the longest
spurs are indeed the most derived in the tree. The available
phylogenies of the genus Aconitum (Utelli et al., 2000; Kita
and Ito, 2000; Luo et al., 2005) are not resolved enough to
detect any lengthening of spurs in the course of evolution.
The pattern of association between spur length and pollinator type in Delphinium and Aconitum (Table 2) is very similar
to the one described in Aquilegia (Whittall and Hodges, 2007):
Delphinium species with the shortest nectar spurs are pollinated by bees or bumblebees, those with intermediate nectar
spurs by hummingbirds, and those with the longest nectar
spurs by hawkmoths. In Delphinium, flat petals act also as pollinator filters and/or attractors, in addition to the spurred petals.
They are used as a visual cue for pollinators due to the hairs
that turn yellow at maturity. Apart from bumblebees that use
them as a landing platform (Laverty, 1980), these petals probably prevent all access to the nectar except to the heaviest and
strongest visitors (Guerrant, 1982).
While successive shifts in function or expression of genes
controlling merism, as well as floral symmetry and organ identity may have played a critical role in the evolution of zygomorphy in Delphinieae, the presence of pollinators able to
exploit the peculiar morphology of the flower has probably
been a key factor for the maintenance and diversification of
this trait.
S U P P L E M E N TARY D ATA
Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following two figures. Fig.
S1. SEM micrographs of the developmental sequence of
Jabbour et al. — Development and evolution of zygomorphy in Delphinieae
Delphinium staphisagria. Fig. S2. SEM micrographs of the
spiral and whorled phyllotaxis in flowers of Ranunculaceae
ACK N OW L E DG E M E N T S
We thank Peter Endress and Hélène Citerne for information
and critically reviewing an earlier version of the manuscript
and Frieda Christie for assistance with the SEM at RBGE.
We also thank two anonymous reviewers for their constructive
comments on the first version of the manuscript. SEM
studies at the Royal Botanic Garden Edinburgh received
support from the SYNTHESYS Project http://www.synthesys
.info/ which is financed by European Community Research
Infrastructure Action under the FP6 ‘Structuring the
European Research Area’ programme. This work received
financial support from the Agence Nationale de la Recherche
no. ANR-07-BLAN-0112-02. F.J. was supported by a fellowship from the Ministère de l’Enseignement Supérieur et de la
Recherche, France.
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