Botanical Journal of the Linnean Society, 2011, 166, 431–443. With 6 figures
Floral organogenesis of Helleborus thibetanus and
Nigella damascena (Ranunculaceae) and its
systematic significance
LIANG ZHAO1, PING LIU1, XIAO-FEN CHE1, WEI WANG2 and YI REN1*
1
College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy
of Sciences, Xiangshan, Beijing 100093, China
2
Received 18 December 2010; revised 5 March 2011; accepted for publication 25 March 2011
Floral organogenesis in Helleborus thibetanus and Nigella damascena was examined and compared using scanning
electron microscopy and light microscopy, and the putative relationships of Helleborus and Nigella were analysed.
H. thibetanus and N. damascena share some features of floral phyllotaxis and development of the sepals, petals,
stamens and carpels, which are also found in other members of Ranunculaceae. However, they differ strongly in
the number and degree of fusion of the carpels: in H. thibetanus, the two carpels are slightly united at the base,
whereas, in N. damascena, the gynoecium is syncarpous and the five carpels are united throughout the ovary.
Differences are also noted in petal development. The blade of the young petal of H. thibetanus develops two bulges
which become connate and then fuse with the blade at the sides, developing more quickly than the blade and
forming a tubular petal. In N. damascena, a single ridge is formed on the petal blade which develops into the
smaller adaxial labium of the bilabiate petal, whereas the blade itself develops into the larger abaxial labium
bearing two pseudonectaries. The outermost stamens are delayed in development in Helleborus, but not in Nigella.
Although the results from our investigation are preliminary, differences in floral development characters suggest
that Helleborus and Nigella may not be closely related and possibly support placement into separate tribes.
© 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166, 431–443.
ADDITIONAL KEYWORDS: floral development – floral morphology – Helleborus – Nigella – Ranunculaceae.
INTRODUCTION
The genus Helleborus L. consists of 20 species and is
distributed throughout southern Europe, northern
Africa, southwestern Asia and western China
(Tamura, 1995). Nigella L. comprises 16 species and
occurs throughout the Mediterranean region and
southwestern Asia, extending westwards to central
Europe and eastwards to central Asia (Zohary, 1983;
Tamura, 1995).
Helleborus and Nigella exhibit some characteristics
which are uncommon in Ranunculaceae. The carpels
are connate only at the base in Helleborus (and also in
Dichocarpum W.T.Wang & Hsiao), but are connate to
*Corresponding author. E-mail: [email protected]
the apex of the ovaries in Nigella. The petals are
tubular or cupular in Helleborus, but bilabiate in
Nigella (and also in Eranthis Salisb.). The sepals are
persistent in Helleborus, but caducous in Nigella
(caducous sepals are common in the family) (Wang,
1979; Tamura, 1995). Both genera have R-type chromosomes. The basic chromosome number is N = 8
(2n = 32) in Helleborus, which is common in the
family, and N = 6 (2n = 12) in Nigella, the lowest
number in the family (Langlet, 1932).
Because of the special features of Helleborus and
Nigella, their systematic position was, and still is,
uncertain. Hutchinson (1959, 1969) treated Helleborus and Nigella as members of Helleboraceae. Most
authors, however, have agreed that they belong to
Ranunculaceae, but some have placed them in different subfamilies (Helleboroideae and Trollioideae; see
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Takhtajan, 1997) or, more commonly, both in Ranunculoideae, either in the same tribe (Helleboreae;
Tamura, 1966; Wang, 1979; Loconte, Campbell &
Stevenson, 1995) or in two tribes (Helleboreae and
Nigelleae; Langlet, 1932; Duncan & Keener, 1991;
Tamura, 1993, 1995). Jensen et al. (1995) placed
Helleborus in its own tribe Helleboreae and placed
Nigella in Delphinieae, based on the integration of
four independent molecular datasets (Hoot, 1995;
Jensen, 1995; Johansson, 1995; Kosuge et al., 1995).
In contrast, Wang et al. (2009) assigned both Helleborus and Nigella tribal rank. Therefore, the relationships and affinities of these genera in Ranunculoideae
remain unclear (Sun, McLewin & Fay, 2001).
The general floral organogenesis of Helleborus and
Nigella was first addressed by Hirmer (1931) and
Schöffel (1932). Detailed descriptions of the mature
flowers of Helleborus and Nigella have been provided,
especially for petals and carpels (Troll, 1933; Hiepko,
1965; Tamura, 1965, 1984; Rohweder, 1967; Kaussmann & Neitzel, 1972; Lang, 1977; van Heel, 1981;
Kosuge & Tamura, 1989; Weber, 1993; Ronse De
Craene & Smets, 1995; Erbar, Kusma & Leins, 1998;
Vesprini, Nepi & Pascini, 1999; Endress & Matthews,
2006; Jabbour et al., 2009). Studies of the floral morphology and development of other genera and infrafamilial taxa of Ranunculaceae have also been
conducted (Lehmann & Sattler, 1994; Chang, Ren &
Lu, 2005; Tucker & Hodges, 2005; Song, Tian & Ren,
2007; Jabbour et al., 2009; Ren et al., 2009; Ren,
Chang & Endress, 2010) and have contributed significantly to a better understanding of the evolution of
Ranunculaceae. A comprehensive comparative study
of the floral organogenesis of Helleborus and Nigella
is still lacking. Here, we present our comparative
study of the floral organogenesis of Helleborus thibetanus Franch. and Nigella damascena L.
MATERIAL AND METHODS
Flower buds of H. thibetanus were collected at all
stages of development between 2003 and 2005 at
Haopingsi, Mt. Taibaishan, Meixian County, Shaanxi
Province, China (altitude, 1100 m; voucher: Bai
Gen-lu 2004036, SANU). Flowers buds of N. damascena were collected between 2007 and 2008 in a
glasshouse at the College of Life Sciences, Shaanxi
Normal University, Xi’an, China (voucher: Wang
Zi-fen 2008001, SANU). The material was fixed in
formalin–acetic acid–ethanol–water (10 : 5 : 50 : 35).
The material was dehydrated in an ethanol and
iso-amyl acetate series, critical point dried in liquid
CO2, mounted on aluminium stubs and viewed in a
scanning electron microscope (Hitachi S-4800). For
light microscopy, the samples were dehydrated in an
ethanol series, infiltrated with xylene and embedded in
paraffin wax. The embedded samples were sectioned
at 8 mm using a Leica RM2235 rotary microtome.
Mounted sections were stained with safranin and fast
green, and viewed under a Leica DM5000B optical
microscope. Photographs of mature flowers were taken
with a Nikon Coolpix 990 digital camera (Fig. 1A, B).
The description of the floral morphology is based on 15
mature flowers. The symbols used in the floral diagrams and floral formulae follow Ronse De Craene
(2010) and Prenner, Bateman & Rudall (2010).
HELLEBORUS
RESULTS
THIBETANUS (FIGS 1A, C, 2–4)
The mature flower is 3.5–6.0 cm in diameter, terminal
and solitary in position, bisexual and polysymmetric.
The calyx consists of five (rarely six) sepals which are
narrow-elliptic, persistent, petaloid and white with
pink–red venation at anthesis, but green when fruiting. The corolla consists of eight to ten petals, which
are small, yellowish-green, tubular, shortly stalked
and nectariferous. There are 15–35 stamens and two
(rarely one) carpels (Fig. 1A, C). Each carpel contains
approximately 10–15 ovules.
The flower bud is subtended by two bracteoles
(Fig. 2A). The sepals, petals and stamens are initiated
clockwise or counterclockwise in a spiral sequence,
with an average divergence angle of 137° between two
consecutively initiated primordia (Fig. 2A–C). The
five (rarely six) sepal primordia are crescent-shaped
and truncate (Fig. 2A, B). There is a relatively long
plastochron between the initiation of the last sepal
and the first petal primordium (Fig. 2C). The petal
and stamen primordia are narrow and hemispherical
during early development (Fig. 2D). Therefore, distinguishing between the last petal and the first stamen
in early development is difficult (Fig. 2D, E). Two
carpel primordia are initiated one after another in an
almost opposite position (Fig. 2G, H). The carpels do
not occupy the entire floral apex, so a small residual
floral apex remains (Figs 2H, 3E, F). This is later
hidden by the accrescent carpels. If only one carpel
primordium emerges, the residual floral apex remains
larger (not shown).
In later development, the sepals enlarge and
enclose all the following floral organs (Fig. 2I). The
development of the petals is delayed and they remain
smaller than the stamens until anthesis (Fig. 2H–J).
Each young petal differentiates into a short stalk and
a large lamina, which is slightly concave (Fig. 2K).
During this stage of development, a depression
appears on the ventral side of the lower part of the
lamina. Then, two median bulges arise in a short
succession at the base of the depression (Fig. 2L). The
bulges expand laterally (Fig. 2M), which results in
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Figure 1. Mature flowers, floral diagrams and floral formula. A, C, Helleborus thibetanus. B, D, Nigella damascena.
Black dots indicate floral axes, black arcs indicate sepals, grey arcs indicate petals, four close circles indicate stamens and
triangles indicate ovaries. The floral diagrams correspond to the floral structures of each species, and the floral formulae
correspond to the genus and thus include the whole range of variation in the number of organs. Scale bars, 1.0 cm.
them fusing with each other and with the lamina at
the margin (Fig. 3A). The fused part grows more
quickly than the lamina and the petal is initially
obliquely cup-shaped (Fig. 3B) and then cup-shaped
(Fig. 3C). Finally, because of the faster growth of the
cupular part, the petal becomes tubular with a short
stalk (Fig. 3D).
The stamens differentiate into filaments and
anthers after the carpel primordia have appeared (Fig. 2H, I). Histological observations show
that the stamen maturation is in a centripetal
direction (Fig. 4A–E), even though the outermost
stamen (St1) is shorter than the other stamens
(Fig. 2I, J).
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Figure 2. Floral organogenesis of Helleborus thibetanus. A, B, Initiation of wide, crescent-shaped and truncate sepals,
with the flower bud subtended by two bracteoles. A, Emergence of the first to third sepal. B, Emergence of the fourth to
fifth sepal. C, Initiation of five petals, showing that the last sepal is much larger than the first petal. D, Initiation of petals
and/or stamens, showing the difficulties in distinguishing the primordia of petals and stamens (arrows). E, Same
developmental stage as in D, side view. F, Number of stamens increased. G, Initiation of the first carpel. H, Two almost
oppositely initiated carpels; star between carpels indicates residual floral apex, petals delayed. I, Outermost stamens
delayed. J, Almost mature flower. K–M, Petal development. K, Petal flattened. L, Appearance of two bulges (arrows).
M, Slightly later stage, bulges (arrows) enlarged. B, bracteole; C, carpel; P, petal; S, sepal; St, stamen. Figures after the
letters indicate the initiation sequence. Scale bars: A–I, K–M, 100 mm; J, 1 mm.
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After the initiation of the carpels, a longitudinal
concavity appears on the ventral side of each carpel
(Fig. 3E). With carpel enlargement, the concavity
becomes deeper (Fig. 3F), and then the sides of the
carpel fuse gradually from the base to the apex,
except for the uppermost part (Fig. 3H). After closure
of the carpels, the growth of the ovary is faster than
that of the upper part of the carpel (Fig. 3I). Subsequently, the style elongates (Fig. 3J). During the
development of the carpels, the residual floral apex
remains and unites the two carpels basally to a slight
degree (Fig. 3G–I). The style becomes slightly reflexed
backwards and everted distally. A short decurrent
stigma differentiates along the ventral slit and at the
tip of the carpel. In the mature flower, it appears
covered with unicellular papillae (Fig. 3K).
NIGELLA
DAMASCENA
(FIGS 1B, D, 5, 6)
The mature flower is 2.5–3.0 cm in diameter, terminal
and solitary in position, bisexual and polysymmetric.
There are five (to eight), petaloid, blue sepals. The
petals are small, blue, numbering five (rarely up to
eight), or absent in some cultivated individuals. Each
petal is stalked and has an abaxial bilobed lamina
with a conspicuous pseudonectary on each lobe and
an adaxial scale. The androecium consists of 30–45
stamens which differentiate into filament and anther.
The carpel number ranges from three to five, with
fused ovaries and free styles (Fig. 1B, D).
The five sepal primordia are initiated spirally and
are crescent-shaped and truncate initially (Fig. 5A).
Some of them then become rounded at the apex
(Fig. 5A–C). Apparently, there is a relatively long
plastochron between the initiation of the last sepal
and the first petal, because initially the last sepal is
much larger than the first petal (Fig. 5A). The petal
primordia are initiated in the same sequence as the
sepals and are rounded (Fig. 5A, B). The stamen
primordia follow the same sequence of the perianth.
They closely resemble petal primordia, and so distinction between the petals and the stamens in
early flower development is difficult (Fig. 5C, D),
and the prediction of whether or not the flower will
have petals is impossible. The divergence angles in
the perianth and stamens fluctuate at approximately 137°, which indicates spiral phyllotaxis
according to the Fibonacci pattern. The carpel primordia are initiated spirally, but their sizes differ
between the individual primordia. The carpel primordia are wider than the stamen primordia
(Fig. 5F). They soon form a whorl and become equal
in size (Fig. 6F). Sets of five, eight and 13 contact
parastichies were recorded in all flowers, despite the
occasional intercalation of isolated stamens between
the contact parastichies (Fig. 5G). After the carpel
primordia are initiated, a small residual floral apex
remains (Figs 5F, G, 6F).
In later developmental stages, the young sepals
enlarge and enclose all the other floral organs. The
young petals start to flatten after the appearance of
the carpels and can be clearly distinguished from the
stamens (Fig. 5H). However, the petals show distinctly delayed development. They remain smaller
than the stamens until anthesis (Fig. 5I). The petals
differentiate into a wide lamina and a narrow base; a
depression appears near the base of the lamina on the
ventral side, and soon a bulge appears at the base of
the depression (Fig. 6A). In subsequent development,
the bulge expands (Fig. 6B) to form a scale (Fig. 6C)
and finally develops into the adaxial labium (Fig. 6D).
The narrow base of the young petal elongates and
finally forms the stalk of the petal (Fig. 6B–E). Meanwhile, the lamina expands, becomes bilobate (Fig. 6B,
C) and forms the abaxial labium (Fig. 6D). A bulge
appears at the base of each lobe (Fig. 6B) and then
develops into the pseudonectary (Fig. 6C, D). Hairs
are present around the two pseudonectaries and the
lower edge of the abaxial labium (Fig. 6C–E).
Each young stamen quickly differentiates into a
filament and anther (Fig. 5H). Histological observations indicate that anther maturation is in a centripetal direction (not documented here).
After the carpels have formed a whorl, a depression
appears at the base of each carpel (Fig. 6G). Subsequently, the flanks of two neighbouring carpels fuse
gradually (Fig. 6H, I). Each of the carpels becomes
horseshoe-shaped (Fig. 6I). The carpels enlarge
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(Fig. 6J) and the remaining free part of each carpel
elongates to form a style (Fig. 6K, L). Before anthesis,
unicellular papillae appear along the margin of the
style to form the stigmatic tissue, and the upper parts
of the styles are twisted (Fig. 6M, N).
DISCUSSION
ASPECTS
OF FLORAL DEVELOPMENT
The floral phyllotaxis of Helleborus and Nigella was
previously considered to be spiral throughout the
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Figure 3. Floral development of Helleborus thibetanus. A–D, Petal development. A, Two bulges fuse with each other and
with the lamina at the sides. B, The fused bulges grow faster than the lamina, and the petal becomes obliquely
cup-shaped. C, Petal becomes cup-shaped. D, Petal becomes tubular. E–K, Carpel development. E, Horseshoe-shaped
carpels; a longitudinal cavity appears. F, Carpels become conduplicate. G, Same developmental stage as F, side view; star
indicates residual floral apex between the two carpels. H, Carpel enlarges and closes. I, Same developmental stage as H,
side view; star indicates residual floral apex between the two carpels. J, Carpels before anthesis. K, Close-up of distal part
of a carpel shown in J, with slightly decurrent and unicellular papillate stigma. C, carpel; P, petal; S, sepal; St, stamen.
Scale bars: A, G, K, 150 mm; I, 200 mm, E, F, 200 mm; B, C, H, 250 mm; D, 1.4 mm; J, 1.5 mm.
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Figure 4. Helleborus thibetanus, longitudinal sections of stamens, showing that the stamen maturation is in a centripetal direction. A, Longitudinal section of flower. B–E, Close-up of different stamens shown in A. B, The outermost stamen
(St1) comprising mature pollen. C, The second stamen (St2), showing microspores in tetrad before microspore release from
tetrad. D, The third stamen (St3), showing microsporogenesis in tetrad. E, The innermost stamen (St4), showing
microsporogenesis in stages of meiosis. C, carpel; S, sepal; St, stamen. Scale bars: A, 1.5 mm; B–E, 5 mm.
flower (Schöffel, 1932). However, from our observations of H. thibetanus and N. damascena, the floral
phyllotaxis of sepals, petals and stamens is spiral, but
that of the carpels is whorled, although they are
initiated asynchronously.
Erbar et al. (1998) found that the divergence angle
between the primordia of the last sepal and the first
petal is approximately 50°, instead of 137°, in H.
foetidus L. However, we found an equal 137° divergence angle between the primordia of the last sepal
and the first petal in H. thibetanus. When we com-
pared our findings with the images of H. foetidus of
Erbar et al. (1998: fig. 1), we found that the initial two
petals were hidden by the developing sepals. This
may suggest a possible explanation for the difference
between the two species in the same genus.
Anther maturation follows the stamen initiation
sequence and is thus centripetal in H. thibetanus and
N. damascena, as in most Ranunculaceae, e.g. Caltha
L and Trollius L. (Song et al., 2007), Adonis L. and
Callianthemum C.A.Mey. (Ren et al., 2009) and
Clematis L. (Ren et al., 2010). However, in Helleborus,
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Figure 5. Floral organogenesis of Nigella damascena. A, The five sepals and the first petal are initiated, showing that
the sepal primordia are crescent-shaped and truncate; the petal primordium is hemispherical and rounded. B, Initiation
of petals. C, Initiation of petals and/or stamens. D, Same as C, side view. E, Initiation of stamens. F, Carpels initiated
spirally and showing size differences. G, Floral organs are arranged in parastichies. H, Delayed petals, side view. I, A later
stage than H, showing centripetal development of stamens and delayed petals. C, carpel; P, petal; S, sepal; St, stamen.
Numbers after letters indicate initiation sequence. Scale bars: B, D, E, G–I, 100 mm; A, C, 120 mm; F, 150 mm.
the delayed elongation of the filament in the outermost stamens gives the impression that the maturation of the stamens is opposite to the direction of their
initiation, and is thus centrifugal (see Aquilegia L.;
Tepfer, 1953; Feng et al., 1995). Some reports of
Anemone indicate that anther maturation can be bidirectional (Chang et al., 2005; Ren et al., 2010). The
developmental pattern of the androecium in H. thi-
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HELLEBORUS & NIGELLA, FLORAL DEVELOPMENT
betanus is thus so far unique in Ranunculaceae.
Further comparative research in other genera of the
family is necessary to gain a better understanding of
this process and of the discordance between the direction of stamen initiation and maturation.
Syncarpy is rare in Ranunculaceae (Endress &
Igersheim, 1999). The carpels have been reported to
be basally connate in Helleborus (Rohweder, 1967;
Wang, 1979; Tamura, 1995) or connate in Nigella
(Toxopéus, 1928; Eames, 1931; Troll, 1933; Kaussmann & Neitzel, 1972; Lang, 1977; Tamura, 1995).
The carpels in H. thibetanus are slightly united at the
base. The present observations in N. damascena are
in agreement with previous studies.
The styles of Nigella are functionally elaborate
structures, first described by Sprengel (1793), and
later documented cinematographically by Weber
(1992, 1993, 1995). At the final developmental stages
of the androecium (pollen release), the styles curve
downward and twist in a corkscrew fashion. This
ensures that, during stigma receptivity, the stigmatic
crest will make contact with the dorsal side of the
pollinator, and thus promote cross-fertilization. In
situations in which cross-fertilization has not
occurred or insects are absent, self-pollination will
take place as a result of a spiral downward movement
of the styles onto the stamens (Toxopéus, 1928).
PETALS
The most variable organs in the flowers of Ranunculaceae are the so-called petals, which are inserted
between the petaloid sepals and stamens and are one
of the most important diagnostic characters within
the family (Tamura, 1995). As the petals of most
genera are nectariferous, they were called in the
German literature ‘Honigblätter’ (honey-phyllaries)
(Prantl, 1887), ‘Nektarblätter’ (nectar-phyllaries)
(Janchen, 1949) or nectary organs (Erbar et al., 1998).
Prantl (1887) presumed that the petals in Ranunculaceae are staminal in origin, and this interpretation
was widely adopted (Hiepko, 1965; Tamura, 1984,
1993; Kosuge & Tamura, 1989; Weber, 1993; Kosuge,
1994; Lehmann & Sattler, 1994; Endress, 1995; Feng
et al., 1995; Erbar et al., 1998; Kramer, Di Stilio &
Schlüter, 2003; Tucker & Hodges, 2005; Gu & Ren,
2007; Song et al., 2007; Ren et al., 2009, 2010). The
present observations of the floral organogenesis of H.
thibetanus and N. damascena and scanning electron
microscopy studies in some other genera of the family
(Kosuge & Tamura, 1988, 1989; Kosuge, 1994;
Lehmann & Sattler, 1994; Endress, 1995; Feng et al.,
1995; Erbar et al., 1998; Tucker & Hodges, 2005; Gu
& Ren, 2007; Song et al., 2007; Ren et al., 2009, 2010)
clearly show a relatively long plastochron between
the last initiated sepal and the first petal, but shorter
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and equal plastochrons between the primordia of the
petals and stamen. In addition, the sepal primordia
are crescent-shaped and truncated, whereas those of
the petals and stamens are hemispherical and
rounded and similar in the early stages of development. These features support the possible evolutionary relationship between the petals and stamens in
Ranunculaceae.
During early developmental stages, each of the
petal primordia of H. thibetanus and N. damascena
differentiates into a short basal stalk and a larger,
emarginate blade. With the enlargement of the blade,
a depression appears at the ventral base of the blade,
and two bulges in H. thibetanus and a transversal
ridge in N. damascena appear at the base of the
depression. This pattern of development was observed
in the genera with petals (Kosuge & Tamura, 1988,
1989; Erbar et al., 1998; Tucker & Hodges, 2005; Ren
et al., 2009), with the exception of Adonis (Kosuge &
Tamura, 1989; Ren et al., 2009), Anemonopsis Siebold
& Zucc. (Kosuge & Tamura, 1989), Coptis Salisb. and
Xanthorhiza Marshall (Kosuge & Tamura, 1989). The
depression and the appendage on the ventral side of
the petal add to the morphological variability found in
the petals of Ranunculaceae.
Based on ontogenetic observations of the petals of
Aconitum napellus L., Aquilegia vulgaris L., H. foetidus and Ranunculus ficaria L., Erbar et al. (1998)
presumed that the two ventral bulges (the appendage) could be considered to be rudimentary (sterile)
adaxial pollen sacs. However, this presumption
cannot explain the single transverse ridge that
appears on the petals of Nigella (this study), Cimicifuga Wernisch [= Actaea L.], Delphinium L., Consolida Gray, Ranunculus L. and some species of
Dichocarpum (Kosuge & Tamura, 1989). If the presumption is correct, the blade portion of the petal
primordium may result from the development of the
two sterile abaxial pollen sacs and/or the connective
tissue. However, this pattern of development was not
found in our examination of Helleborus, Nigella or in
previous investigations of other genera (Kosuge &
Tamura, 1988, 1989; Lehmann & Sattler, 1994;
Tucker & Hodges, 2005; Song et al., 2007; Jabbour
et al., 2009; Ren et al., 2009; this study). On the basis
of the later development of the two bulges and the
transverse ridge in the flowers of Helleborus and
Nigella, we suggest that the developmental result of
the appendage may be a highly modified structural
and functional feature for concealing the nectar and
controlling the access of the visitors (see Erbar et al.,
1998).
RELATIONSHIPS
OF
HELLEBORUS
AND
NIGELLA
Molecular systematics studies have inferred that
Helleborus forms a sister relationship to Caltha
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Figure 6. Floral organogenesis of Nigella damascena. A–E, Petal development. A, Depression on ventral base of the blade
of the petal. B, Ridge enlarges into a scale and petal becomes bilabiate, arrows indicate pseudonectaries. C, A later stage
of petal development. D, E, A petal before flower anthesis. D, Ventral view, arrows indicate position of two pseudonectaries. E, Side view, arrow indicates a cavity in which the nectar is secreted. F–N, Carpel development. F, Carpels
arranged in a whorl but different in size. G, A small depression appears in the centre of each carpel. H, Slightly later
stage, carpels fuse at the flanks. I, Carpels enlarge and become conduplicate and horseshoe-shaped. J, A later stage. K,
Apical part of one of the carpels removed. L, Close-up of upper part of carpels. M, N, Close-up of upper part of ventral
slit of carpel. M, Stage before appearance of stigmatic tissue. N, Mature stigmatic tissue. C, carpel; St, stamen. Scale bars,
100 mm.
䉳
(Hoot, 1995) or to tribe Cimicifugeae (Ro, Keener &
McPheron, 1997; Hoot, Kramer & Arroyoy, 2008).
Alternatively, Jensen et al. (1995) and Wang et al.
(2009) have suggested that Helleborus forms no close
relationships with other taxa. Comparison of Helleborus with tribe Cimicifugeae is difficult because of
the lack of descriptions of floral development. The
petals are absent and the carpels are free in Caltha,
which differs from Helleborus (Song et al., 2007).
Molecular systematics studies have suggested that
Nigella is sister to Delphinieae (Jensen et al., 1995;
Hoot et al., 2008). Jabbour et al. (2009: fig. 8) proposed that, if the Nigella morph (i.e. individuals with
eight petals) is ancestral, the corolla of Delphinieae
may result from a reduction of four petals (as present
in Delphinium, with two spurred, two nonspurred and
four rudimentary petals) or six petals (Aconitum, with
two spurred and six rudimentary petals), in conjunction with the differentiation of partially new petal
identities. Based on our observations of the floral
ontogeny, we could not find any relationship between
N. damascena and tribe Delphinieae, because the
number of petals is inconsistent, with mostly five to
six and rarely eight. Nonetheless, the floral development features of Nigella and tribe Delphinieae are
more similar than those of other genera (see Feng
et al., 1995; Tucker & Hodges, 2005; Gu & Ren, 2007;
Song et al., 2007; Ren et al., 2009, 2010). In Nigella
and Delphinieae, the sepals, petals and stamens
develop spirally, whereas the carpels are whorled.
Although Jabbour et al. (2009) did not observe this
floral developmental feature, the whorled carpels are
clearly apparent in the authors’ images (fig. 3K for
Aconitum napellus; fig. 5E for Delphinium grandiflorum L.; fig. S1B for D. staphisagria L.).
Helleborus and Nigella were, at one time, considered to be closely related and were included in the
same tribe Helleboreae (Langlet, 1932; Hutchinson,
1959, 1969; Tamura, 1966; Wang, 1979; Loconte et al.,
1995). In contrast, other authors have placed them in
different tribes (Duncan & Keener, 1991; Tamura,
1993, 1995; Jensen et al., 1995; Wang et al., 2009).
Helleborus thibetanus and N. damascena share
several floral organogenetic features: (1) spiral initiation of the sepals, petals and stamens; (2) broad,
crescent-shaped and truncate sepal primordia; (3)
narrow, hemispherical and rounded petal and stamen
primordia; (4) a relatively long plastochron between
the last sepal and the first petal; (5) delayed development of the petals; (6) centripetal stamen initiation
and maturation; and (7) horseshoe-shaped young
carpels. However, these features are common in
Ranunculaceae (Schöffel, 1932; Hiepko, 1965; Kosuge
& Tamura, 1989; Erbar et al., 1998; Chang et al.,
2005; Tucker & Hodges, 2005; Gu & Ren, 2007; Song
et al., 2007; Jabbour et al., 2009; Ren et al., 2009,
2010). In our examination of the floral organogenesis
of H. thibetanus and N. damascena, we note the
following floral developmental features that differ
between the two species: (1) the carpels are only
slightly united at the base in H. thibetanus, whereas,
in N. damascena, the gynoecium is syncarpous and
the carpels are united throughout the ovary; (2) the
appendage at the ventral base of the blade of the
young petal is formed by two bulges in H. thibetanus
instead of a ridge in N. damascena, and their development also differ; and (3) the outermost stamens are
delayed in growth in H. thibetanus, but not in N.
damascena. The differences observed in the floral
developmental features of these two species support
Helleborus and Nigella being placed in separate
tribes. However, further investigations of several
species within each genus are necessary to support
this conclusion.
ACKNOWLEDGEMENTS
We sincerely thank Professor Peter K. Endress (University of Zurich), Dr Julien B. Bachelier and Robert
L. Baker (Harvard University), Mare Nazaire (Washington State University), Dr Xin-Wei Li (Wuhan
Botanical Garden, Chinese Academy of Sciences), Dr
Xiao-Hui Zhang (Shaanxi Normal University) and Dr
Jeremy Lundholm (Saint Mary’s University) for
helpful comments and for careful reading of the
manuscript. We are very grateful to Mr Yin-Hou Xiao
(Institute of Botany, Chinese Academy of Sciences)
and Mr Yao-Hui Ren (Shaanxi Normal University) for
assistance in scanning electron microscopy. This
© 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166, 431–443
442
L. ZHAO ET AL.
project was supported by grants from the National
Natural Science Foundation of China (Nos. 30870179
and 30800059).
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