Annals of Botany Page 1 of 14

doi:10.1093/aob/mcv177, available online at www.aob.oxfordjournals.org

Space matters: meristem expansion triggers corona formation in Passiflora

Regine Claßen-Bockhoff * and Charlotte Meyer
Institut für Spezielle Botanik, Johannes Gutenberg-Universität Mainz, Germany
* For correspondence. E-mail classenb@uni-mainz.de

Received: 18 August 2015 Returned for revision: 10 September 2015 Accepted: 6 October 2015

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
 Background and aims Flower meristems differ from vegetative meristems in various aspects. One characteristic
is the capacity for ongoing meristem expansion providing space for new structures. Here, corona formation in four
species of Passiflora is investigated to understand the spatio-temporal conditions of its formation and to clarify
homology of the corona elements.
 Methods One bird-pollinated species with a single-rowed tubular corona (Passiflora tulae) and three insect-
pollinated species with three (P. standleyi Killip), four (P. foetida L. ‘Sanctae Martae’) and six (P. foetida L. var.
hispida) ray-shaped corona rows are chosen as representative examples for the study. Flower development is
documented by scanning electron microscopy. Meristem expansion is reconstructed by morphometric data and
correlated with the sequential corona element formation.
 Key results In all species, corona formation starts late in ontogeny after all floral organs have been initiated. It is
closely correlated with meristem expansion. The rows appear with increasing space in centripetal or convergent
sequence.
 Conclusions Based on the concept of fractionation, space induces primordia formation which is a self-regulating
process filling the space completely. Correspondingly, the corona is interpreted as a structure of its own, originating
from the receptacle. Considering the principle capacity of flower meristems to generate novel structures widens the
view and allows new interpretations in combination with molecular, phylogenetic and morphogenetic data.

Key words: Corona, de novo structures, flower meristem, fractionation, meristem expansion, morphogenesis,
Passiflora foetida ‘Sanctae Martae’, P. foetida var. hispida, P. standleyi, P. tulae, self-organization, space.

INTRODUCTION
Flowers are usually defined as short shoots with reproductive
organs. However, their meristems differ from vegetative shoot
apical meristems in being determinate and highly plastic
(Ronse de Craene, 2010; Claßen-Bockhoff, 2015). Differential
meristem activity (Leins and Erbar, 2008) provides flowers
with a broad developmental potential, reflected in the exciting
diversity of flower shapes and functions.
One of the typical flower capacities is represented by the for-
mation of a corona, i.e. an attractive structure located between
the petals and stamens. The well-known examples from the
Amaryllidaceae (Narcissus, Pancratium) have been controver-
sially interpreted within the last 150 years as originating either
from the perigon (Eichler, 1875; Arber, 1937; Chen, 1971;
Singh, 1972) or from stamens (Čelakovský, 1898; Velenovský,
1910; Glück, 1919; Goebel, 1933; Schaeppi, 1939).
Phylogenetic data indicate that the corona evolved several times
in parallel in this lineage, allowing for different morphogenetic
origins (Meerow et al., 1999; Waters et al., 2013).
Less well known yet equally conspicuous is the corona in the
passionflower Passiflora (Passifloraceae). The attractive flow-
ers belong to the most loved ornamental plants in the world.
The first reports date back to the 16th century when the peculiar
shape of the floral structures was interpreted as a symbol of the
passion of Christ, from whence came the name of the flower
(Kugler and King, 2004).
Passiflora is the largest genus in the Passifloraceae, with
>500 species (Krosnick and Freudenstein, 2005). Flowers are
actinomorphic and pentamerous, exposing an androgynophore
with five anthers and three styles (Figs 1 and 2). As nectar is
hidden at the base of the flower, the pollinator moves around
the androgynophore. In young flowers, the anthers come into
contact with the pollinators and load pollen on their head or
back. In older flowers, the stylar arms bend backwards and pol-
len is transferred to the receptive tissue (Janzen, 1968).
The most attractive element of the flower is the corona,
which is highly diverse in the number, shape, size and colour of
its elements. Following the terminoloy of Puri (1948), the com-
plete set of structures includes (in centripetal order) long fila-
mentous structures, called ‘radii’, short structures called ‘pali’,
and the ‘operculum’ which is the bulge-shaped inner border of
the corona (Fig. 1). Together with the ‘limen’, an effiguration
close to the androgynophore, the operculum covers the nectar-
producing tissue, called the annulus.
As in Narcissus, the corona in Passiflora has been controver-
sially interpreted. Lindley (1830), Puri (1947, 1948) and de
Wilde (1974) discussed a morphogenetic origin from the sepals
and petals, while Masters (1871) interpreted the structures as
‘organs sui generis’. From a systematic point of view, Endress
(1996) assumed that the corona elements may correspond to a
group of modified staminodes. However, ontogenetic studies
did not confirm this view (Bernhard, 1999).

C The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com
Page 2 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora

sty

sti

st

ra
p
ag
pa

s
o

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
li
an

FIG. 1. Passiflora. Schematic side view of a typical flower illustrating floral structures. Grey shading, flower cup after full expansion with corona elements and limen.
ag, androgynophore, an, annulus, li, limen, o, operculum, p, petal, pa, pali, ra, radii, s, sepal, st, stamen, sti, stigma, sty, stylus.

A B

C D

FIG. 2. Passion flowers. (A) Passiflora foetida ‘Sanctae Martae’. (B) Passiflora foetida var. hispida. (C) Passiflora standleyi. (D) Passiflora tulae.

In the present paper, the genesis of the corona in passion- sides (Claßen-Bockhoff, 2015). It is assumed that corona for-
flowers is investigated to understand its formation with respect mation is coupled to meristem expansion, providing space for
to space. Given that the flower meristem is determinate with no additional structures. To test this hypothesis, flower develop-
potential for apical growth, it has the capacity to expand on all ment is investigated in four Passiflora species differing in the
 Claßen-Bockhoff and Meyer — Corona formation in Passiflora
number and shapes of their corona elements (Fig. 2). Passiflora
foetida var. hispida and P. foetida ‘Sanctae Martae’ (both sub-
genus Passiflora, sect. Dysosmia) have six and four series of
corona elements, respectively. Passiflora standleyi has three
rows and P. tulae has a single-row tube-like corona (both sub-
genus Decaloba, section Decaloba). The focus of the study is
on meristem conditions, corona initiation and element forma-
tion during flower development. If the flower meristem expands
during primordia production, space may play a significant role
in generating diversity.
MATERIALS AND METHODS
Passiflora foetida var. hispida, P. foetida ‘Sanctae Martae’,
P. standleyi and P. tulae are grown at the Botanical Garden at
Mainz University, Germany. Cuttings originated from the nurs-
ery ‘Blumen & Passiflora’, Witten, Germany. Flowers are
4–6 cm in diameter and pollinated by bees, except P. tulae
which is hummingbird-pollinated (Janzen, 1968).
Buds of different age were picked from the plants and fixed
in 70 % ethanol. Longitudinal dissections were made with razor
blades, which were regularly replaced with new ones. After re-
moving the stamen and carpel primordia, the probes were incu-
bated in 80, 90 and 96 % ethanol, each step for 30 min. After
that, material was transferred to 1:1 ethanol–acetone and then
to 100 % acetone. Critical-point drying was conducted using a
BAL-TEC CPD030 instrument with CO2 as exchange agent.
The probes were sputtered with gold (BAL-TEC SCD005) and
analysed under an XL 30 ESEM scanning electron microscope
(Philipps). Each step was conducted according to the manufac-
turer’s protocol.
In total, 178 buds and flowers were dissected and 145 probes
analysed under the SEM. To correlate meristem expansion with
corona element formation, developmental stages were defined
for each species. The dimensions of at least eight probes per
species and stage were measured in a standardized way using
Imagic ims Client 12v. The results are documented by boxplots
using QtiPlot 65v. As corona development is a continuous pro-
cess, transitional forms appear as extreme values within the de-
fined stages and are indicated by the standard deviation.
RESULTS
Flower formation
The four Passiflora species grow as lianas. The shoot apical
meristem remains vegetative and produces leaf primordia in a
spiral sequence (Fig. 3A: 1–10). Axillary meristems appear at a
few nodes off the apex and start to expand laterally. In P. stand-
leyi, each meristem develops three parts (Fig. 3B, C). The larg-
est part gives rise to the tendril meristem (tm), while the two
lateral parts originate lateral flowers (fm). Each flower is sub-
tended by a bract (fsb) and has two prophylls (pp) (Fig. 3D, E).
Due to intercalary activity, the three bracts are dislocated in a
concaulescent way and finally form an outer calyx below the
flower. At this time, an accessory meristem part appears in the
adaxial–median position (Fig. 3E, F). It produces leaves with
stipules, indicating that it is a vegetative meristem. Indeed, lat-
eral branches originate from the accessory buds in a later onto-
genetic stage.
Page 3 of 14
Flower development is highly similar among the four spe-
cies. In P. foetida ‘Sanctae Martae’ and P. foetida var. hispida,
five sepal primordia appear in a spiral sequence (Fig. 4A).
After meristem expansion, five petal primordia follow in alter-
nate positions (Fig. 4B). Next, five anther primordia appear op-
posite the sepals. They are roundish and in part covered by the
growing petals (Fig. 4C). When the three carpel tips arise, fila-
ments separate from the anthers (Fig. 4D, E: fi). The carpels
continue to grow while the tissue below the petals and sepals
starts to extend and to form a receptacular cup (Fig. 4F, G: cu).
This cup gradually expands with ongoing ovary and anther de-
velopment, providing space between the petals and anthers
(Fig. 4H: arrow). In this space, called the ‘corona insertion
Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
area’ (cia), the first corona elements appear when the pollen
sacs are initiated, and the ovary continues elongating (Fig. 4I,
H). With ongoing anther and style development, cup expansion
continues, allowing more and more corona elements to be
formed (Figs 5–10).
Flower development in P. standleyi and P. tulae only differs
from the described sequence in a different timing of organ de-
velopment. Petal growth is retarded (compare Fig. 4C, K) and
the first corona elements appear later, i.e. after ovary develop-
ment (compare Fig. 4H, L).
Corona development
The four species clearly differ in number and shaping of the
corona elements (Fig. 2). They have in common that the corona
is always formed in a late stage of flower development when
space is made available by meristem expansion.
Passiflora foetida ‘Sanctae Martae’. Corona development in P.
foetida ‘Sanctae Martae’ passes through four stages which are
each defined by the formation of a new structure. The starting
point is the cia, which appears as a flat bulge on the inner side
of the receptacular cup between petal and anther primordia
(Fig. 5A).
Stage 1. The cia spans about 100 mm (Table 1). The first radii
appear as small effigurations at its distal border (Fig. 5B).
Stage 2. The operculum appears (Fig. 5C: arrow). When the
cup is >150 mm extended, the proximal border of the corona
tissue arches upwards. Thereby, the cia becomes clearly sepa-
rated from the remaining tissue. Below the protruding opercu-
lum, a narrow ring-shaped hollow is formed. At the same time,
the first radii of the second row appear.
Stage 3. The second series of radii (ra) is completed. The cia
measures about 250 mm (Fig. 5D; Table 1).
Stage 4. The two series of pali appear simultaneously; the cia
and operculum continue growth (Fig. 5E). The meristem tissue
between the operculum and the base of the stamens doubles its
size (from approx. 20 mm to >200 mm; Fig. 6A, B: o, st). The li-
men (li) appears as an outgrowth (Fig. 6B, C). As soon as the
androgynophor (ag) elongates and provides further space at the
base of the flower, the limen expands in the vertical direction
(Fig. 6C, D: li). Its tip turns inwards and becomes covered by
the hook-shaped bent operculum (Fig. 6D). Both structures
cover the nectar, which originates from the nectar-producing
tissue (annulus) below the operculum (Fig. 5F: arrow).
When all elements of the corona are formed, the height of
the flower cup is about 340 mm (Table 1). The cia has expanded
Page 4 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora

A B
fm fm
tm
stp
7 10
l
2
4
5
sam
9
C
1

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
stp
6 3
8 fsb
fsb
I fm fm

D E
pp
pp
s s
fsb s s

pp
pp asm pp
fsb
fsb
tm
stp
F

l l

stp

FIG. 3. Shoot development in Passiflora standleyi. (A) The shoot apical meristem (sam) segregates leaves (l) with stipules (stp) in a spiral sequence. 1–10, down-
wards sequence of lateral primordia and axillary meristems. (B–D) The axillary meristem merges into the tendril meristem (tm) and forms two lateral meristems giv-
ing rise to subtending bracts (fsb), prophylls (pp) and flower meristems (fm). (E) When sepals (s) arise, an accessory meristem part (asm) appears in the adaxial
median position. (F) This produces stipulated leaves, indicating its vegetative nature. Scale bar ¼ 200 mm.

4-fold since the first fractionations (Fig. 7A). At anthesis, the
radii are 2 cm long, filamentous structures, which are bent back-
wards and increase the attractiveness of the flower (Fig. 2A).
The pali remain much shorter (approx. 05 cm) and surround
the androgynophore.
Passiflora foetida var. hispida. While P. foetida ‘Sanctae
Martae’ has four corona series (two radii, two pali), P. foetida
var. hispida forms six rows, two series of radii and four rows of
pali. The corona develops in five steps (Fig. 7B).
Stage 1. The cia spans about 150 mm (Table 1), i.e. it is about
50 mm larger than that in P. foetida ‘Sanctae Martae’. The outer
radii elements appear (Fig. 8A, B).
Stage 2. The operculum appears as a distinct bulge at the
lower margin (Fig. 8C).
Stage 3. The cia has gradually extended and now measures
>340 mm in height. The second row of radii and the
lowest pali series (row 6) develop almost simultaneously
(Fig. 8D, E). The limen starts to grow and the operculum
continues bulging.
 Claßen-Bockhoff and Meyer — Corona formation in Passiflora Page 5 of 14

A B C
s
s p
s p st
p s p st
s
st p
s s p
s p p st
s
p st p
s

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
D E F
a
c c
c a c
c c

s
fi
p

G H I sti

sty

cu

J K L

s
p
s st p st
st st
st s
p st
st st p st
s st
p s

FIG. 4. Flower development. (A–I) Passiflora foetida ‘Sanctae Martae’ (also representing P. foetida var. hispida). (A) Sepals (s) develop in a spiral sequence. (B)
Petal primordia (p) appear almost simultaneously followed by five stamen primordia. (C) Petals elongate and cover the stamen primordia (st); their slightly elevated
insertion area indicates the beginning of flower cup formation. (D, E) When the filaments (fi) start to elongate (arrows), three carpel (c) tips appear in the centre of
the flower. (F) The carpel tips elongate and the cup continues to grow, separating petals from stamens. (G, H) The first corona elements (black arrow) appear at the
inner side of the cup (cu). (I) Styles (sty) and stigmas (sti) differentiate at the tip of the ovary; the androgynophore has not yet developed. (J–L) Passiflora standleyi
(also representing P. tulae). The two species from Passiflora subgenus Decaloba differ in two aspects from flower development in P. foetida: petal growth is retarded
compared with stamen development (J, K) and corona elements appear in a later stage; in stage L, which is comparable with stage H, no corona elements are visible.
Scale bars ¼ 200 mm (A–C, J, K), 500 mm (D–I).
Page 6 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora

A B C

p p

cia x x

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
D E

ra
o

li

FIG. 5. Corona development in P. foetida ‘Sanctae Martae’ (androecium and gynoecium removed). (A, B) Stage 1: the expanding receptacle forms a bulge (cia) at its
inner side; first radii appear at its upper rim. (C) Stage 2: the operculum (arrow) forms the lower rim of the developing corona (st age 2). (D) Stage 3: the second row
of radii (ra) is completed; the operculum (o) is distinct. (E) Stage 4: two rows of pali appear almost simultaneously. (F) All elements of the corona are formed; the
operculum and limen cover the nectar-secreting annulus (arrow). All images are at the same scale. Scale bar ¼ 500 mm.
 Claßen-Bockhoff and Meyer — Corona formation in Passiflora Page 7 of 14

TABLE 1. Corona meristem expansion during development (median values and standard deviation)

Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

P. foetida, Sanctae Martae’ (n  12) 1055 6 121 1569 6 160 2530 6 32,3 3391 6 465
P. foetida var. hispida (n  10) 1457 6 306 2405 6 418 3419 6 231 3996 6 358 5174 6 828
P. standleyi (n  8) 1044 6 112 1499 6 111 2195 6 319 3091 6 285 3481 6 526
P. tulae (n  9) 970 6 128 1761 6 222 2527 6 143 3326 6 331

The stages are species specifically defined and not directly comparable.
n, number of samples; bold, compare with Fig. 11.

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
A B
p

st
co
st

o
o

C D

co
st st
co

o
o
ag ag
li
li

FIG. 6. Limen formation in P. foetida ‘Sanctae Martae’. (A, B) When the receptacle widens, space is provided between the developing operculum (o) and the stamen (st)
base; below the operculum a ring-shaped hollow is formed (white arrow). The limen originates as an emergence of the receptacle (B: black arrow; co, corona). (C, D)
When the androgynophor (ag) elevates the stamen base, further space is provided. Operculum and limen enlarge and form the nectar chamber. Scale bar ¼ 200 mm.
Page 8 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora

A 700 B
400
600
Primordium size (μm)

Primordium size (μm)

350

300 500

250 400

200 300

150 200

100 100

1 2 3 4 1 2 3 4 5

500
C 450 D

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
450
400
Primordium size (μm)

Primordium size (μm)

400
350
350
300
300
250
250
200
200
150
150
100
100
1 2 3 4 5 1 2 3 4
Developmental stage Developmental stage

FIG. 7. Temporal relationship between flower cup expansion (primordium size) and developmental stage of the corona. (A) P. foetida ‘Sanctae Martae’. (B) P. foetida
var. hispida. (C) P. standleyi. (D) P. tulae. Stage 1: formation of first radii. Stage 2: formation of the operculum. Stage 3: formation of the second corona series (radii
in A, pali in C), simultaneous formation of the second radii series and forth pali series (B), constriction between radii and operculum (D). Stage 4: simultaneous frac-
tionation of two rows of pali (A), fractionation of third (B) and second series of pali (C) and massive folding of the operculum (D). Stage 5: simultaneous formation
of the two last pali series (B) and massive folding of the operculum (C).

Stage 4. The second pali series (row 5) is formed above the
first one (Fig. 8F). Operculum and limen cover the nectar
chamber (arrow).
Stage 5. The last two pali series (rows 4 and 3) appear almost
simultaneously (Fig. 7G). After completion of all corona ele-
ments, the radii start to elongate (Fig. 7H).
Compared with P. foetida ‘Sanctae Martae’, the cia is much
larger in P. foetida var. hispida (Table 1; Fig. 7A, B). Corona
element formation starts when the cup measures approx.
150 mm, i.e. when it is 15-fold larger than in the former spe-
cies. The total extension of the cia is, at approx. 360 mm, much
greater than in P. foetida ‘Sanctae Martae’ (extension approx.
235 mm). At stage 3, there is space left between the radii and
the operculum (approx. 250 mm) which is gradually covered by
pali elements. Altogether, fractionation in P. foetida var. his-
pida proceeds in a convergent pattern, filling the space with
radii and pali from both margins of the corona.
Passiflora standleyi. Passiflora standleyi has only three rows of
corona elements, one series of radii and two of pali.
Nevertheless, five developmental stages can be distinguished.
Stage 1. The cia is formed. It has a similar size to that in
P. foetida ‘Sanctae Martae’ (approx. 100 mm). Radii appear
(Fig. 9A; Table 1).
Stage 2. The operculum develops at the lower margin of the
cia (Fig. 9B). In contrast to the two previous species, it is struc-
tured from the early beginning.
Stage 3. The upper row of pali appears below the series of ra-
dii (Fig. 9C, D: arrow).
Stage 4. The second (lower) row of pali (Fig. 9E: pa) follows
immediately. The formation of corona elements is now
completed.
Stage 5. In contrast to the previous species, ongoing cia ex-
tension (Table 1; Fig. 7) enlarges the operculum zone. As a
consequence, the operculum becomes the dominant structure of
the corona (Fig. 9F, G). It expands, continues bulging and bend-
ing, and ends up as a massive ring (Fig. 9H). As in the other
species, the radii elongate to conspicuous elements and the pali
remain relatively short.
Passiflora tulae. The corona in P. tulae differs from that in the
other species. No rows of radii and pali are formed. Instead, the
upper margin of the cia grows out and develops a single con-
spicuous tube-like structure (Fig. 2D). Nevertheless, four stages
of development can be recognized.
Stage 1. The cia is formed as a flat ring. Its upper margin
starts growing (Fig. 10A, B).
Stage 2. The operculum separates from the underlying tissue
(Fig. 10C).
Stage 3. The operculum forms a ring-shaped constriction and
becomes clearly separated from the corona tube (Fig. 10D, E).
Stage 4. While the corona elongates continuously, the
operculum starts to grow by bending and folding its surface
(Fig. 10F, G). The limen appears below the operculum only at a
later stage.
 Claßen-Bockhoff and Meyer — Corona formation in Passiflora Page 9 of 14

A B C

p p

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
D E F

2 5
6
cia

G H

34

FIG. 8. Corona development in P. foetida var. hispida (androecium and gynoecium removed). (A, B) Stage 1: formation of the bulge and first radii (p, petal). (C)
Stage 2: development of the operculum (arrow). (D, E) Stage 3: the second row of radii (2) and the lowest row of pali (6) appear simultaneously. (F) Stage 4: the sec-
ond series of pali is formed; the operculum and limen cover the nectar chamber (arrow). (G) Stage 5: the last two series of pali appear simultaneously. (H) The co-
rona is completed and all elements start to elongate. All images are at the same scale. Scale bar ¼ 500 mm.

The cia in P. tulae resembles those of P. foetida ‘Sanctae
Martae’ and P. stanleyi in both initial dimension (approx.
100 mm) and degree of expansion (Table 1). It differs from
them in the size of the first elements, which occupy the whole
cia, leaving no space for further corona elements.
DISCUSSION
Passiflora cleary shows that space matters. This is evident in
the vegetative plant body, but particularly in the flowers.
In the leaf axils, adaxial accessory buds appear after meri-
stem expansion, when the tendril meristems are dislocated to-
wards the abaxial side. Vegetative branching originates from
these accessory buds, while the tendrils give rise to one or two
lateral flowers (Troll, 1938–39).
During flower development, space originates from the expan-
sion of the receptacle giving rise to the corona and limen. First,
the floral organs are initiated using the existing space com-
pletely. Then, the receptacle widens by intercalary meristem ac-
tivity. A floral cup (hypanthium sensu Ronse de Craene, 2010)
Page 10 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora

A B C D

cia

E F

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
ra 1
pa 2

G H

FIG. 9. Corona development in P. standleyi (androecium and gynoecium removed). (A) Stage 1: the corona insertion area (cia) is present and the first radii are formed
(p, petal). (B) Stage 2: operculum appears (arrow). (C, D) Stage 3: the first row of pali is formed (arrow). (E) Stage 4: the second row of pali (pa) appears (ra, radii).
(F–H) The operculum (o) forms a massive ring due to ongoing meristem expansion. All images are at the same scale. Scale bar ¼ 500 mm.

originates, separating the sepals and petals from the anthers.
From the newly generated space, the corona elements appear.
The first step is the formation of an upper rim followed by the
bulging of the operculum. These two structures mark the bor-
ders of the later corona and are separated from each other by
ongoing expansion of the receptacle. The first corona elements
appear in a single row slightly differing in shape and size. With
expansion, new elements appear at the gaps between two previ-
ous elements. The process continues until the whole space is
filled. Outside the corona, additional space is provided at the
base of the flower when the filaments elongate and the andro-
gynophore rises. Ongoing meristem expansion between the co-
rona and androgynophore provides space for the limen and
nectar chamber.
Spatial constraints in expanding meristems
The number of rows depends on the initial size of the cia, the
degree of expansion and the relative size of the corona
elements.
In P. foetida var. hispida, the initial space (approx. 146 mm)
and meristem expansion (approx. 360 mm) are highest among
the species investigated (Table 1). This correlates with the high
number (six) of corona rows. In P. tulae, the initial space
(97 mm) and meristem expansion (approx. 235 mm) are lower
than in the remaining species. However, the tubular shape of
the corona is less the effect of lack of space and more that of
different element proportions. The whole cia is used by the up-
per rim and the massive operculum, leaving no space for
 Claßen-Bockhoff and Meyer — Corona formation in Passiflora Page 11 of 14

A B C

cia

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
D G

E co

o
F

FIG. 10. Corona development in P. tulae (androecium and gynoecium removed). (A, B) Stage 1: the corona insertion area (cia) forms a bulge and separates from the
petals (p) by an upper rim. (C) Stage 2: the operculum (arrow) appears. (D, E) Stage 3: the upper rim starts to elongate and to form the corona tube; the operculum
separates from the tube by a clear constriction (arrow). (F, G) Stage 4: the operculum and tube continue to grow. All images are at the same scale. Scale
bar ¼ 500 mm.

additional elements (Fig. 11D, D’). The rim forms a conspicu-
ous tube guiding the hummingbird’s beak to the nectar. A small
change in the proportion of the corona elements thus allows the
flower to adapt to bird pollination.
As cia and meristem expansion vary among species, a direct
comparison of the spatio-temporal conditions during corona for-
mation is difficult. However, in stage 3 (P. foetida var. hispida),
4 (P. foetida ‘Sanctae Martae’, P. tulae) and 5 (P. stanleyi), the
hypanthial cups have a similar size (approx. 340 mm), allowing
for comparison of relative proportions and relative occupation of
space by the single rows (Fig. 11). At this size, the first series
and the operculum are present in all species. In P. tulae
(Fig. 11D), the space is completely covered by these two struc-
tures, the upper rim giving rise to the corona tube while the
Page 12 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora

A B C D

Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
A′ B′ C′ D′

1 1
1
3
3 3
6
5 4 1
4
4
5 2
3
2 2
2

FIG. 11. Corona element formation as a response to space. (A–D) Corona of the four species at a cup extension of about 340 mm (double arrow, see Table 1). (A’–D’)
Schematic representation of the same developmental stage showing the relative proportion and use of space of the corona elements. Numbers indicate the sequence
of row formation; arrows indicate centripetal (A’, C’) and convergent (B’) direction of fractionation. (A, A’) P. foetida ‘Sanctae Martae’. (B, B’) P. foetida var. his-
pida. (C, C’) P. standleyi. (D, D’) P. tulae.

operculum forms a massive bulge. In the other three species,
space is left for additional corona series, however with different
relative proportions. In P. standleyi (Fig. 11C), the single row of
radii is well developed, leaving space only for two series of pali.
In the two remaining species, a second row of radii is already
present. Due to relative proportions, there is more space left in P.
foetida var. hispida (Fig. 11B) than in P. foetida ‘Sanctae
Martae’ (Fig. 11A), giving rise to four instead of two series of
pali.
Fractionation and sequence of corona element formation
It is evident that new corona elements appear as soon as
space is made available. In fact, newly generated space appears
to stimulate element formation. Thereby, the gaps between ex-
isting elements are filled in an apparently autonomous manner.
Irregular structures are the result of this space-filling process.
Corona element formation can be explained by the process
of fractionation. This process only occurs at determinate meri-
stems (e.g. leaves, flowers) and is defined as the complete use
of the existing space (Claßen-Bockhoff and Bull-Hereñu, 2013;
Claßen-Bockhoff, 2015). Computational models, based on the
concept of directed auxin flow (Reinhardt et al., 2003), clearly
show that primordia can arise due to a self-organizing process
(Runions et al., 2014). Ongoing meristem expansion changes
the spatial conditions and is automatically followed by fraction-
ation. This happens late in ontogeny and results in the forma-
tion of novel structures, i.e. ‘organs sui generis’ sensu Masters
(1871).
As developing meristems maximize the usage of space by
competitive partitioning (Prusinkiewicz and Barbier de Reuille,
2010), fractionation results in diverse patterns. This is also
evident in Passiflora. In P. foetida ‘Sanctae Martae’ and
P. stanleyi, corona elements are initiated in a centripetal direc-
tion, which is consistent with studies in P. edulis and P. quad-
rangularis (both Moncur, 1988), P. holosericea, P. perakensis,
P. molucacana var. glaberrima (all Krosnick et al., 2006) and
P. caerulea (Hemingway et al., 2011). In P. foetida var. his-
pida, in contrast, the three rows of pali are fractionated in the
centrifugal direction. Such a convergent pattern was also
 Claßen-Bockhoff and Meyer — Corona formation in Passiflora
observed in P. racemosa with three rows of corona elements
(Bernhard, 1999) and in P. edulis, P. coccinea and P., Lady
Margret’ (Abizza, 2010). These examples and the fact that
P. edulis obviously shows both patterns illustrate that the cen-
tripetal sequence is not as conserved in Passiflora as assumed
(Hemingway et al., 2011).
The reason for the different fractionation patterns is presently
unknown. It is possible that the sequence of element formation
follows the centripetal or centrifugal expansion of the cia. This
would mean that the direction of mitotic activity within the cia
differs among species. Alternatively, it could be that the mar-
gins of the cia trigger fractionation. In all species, the upper rim
and the operculum appear first, limiting the expanding meri-
stem. Wounding experiments in sunflower meristems have
shown that primordia can arise from artificially induced mar-
gins and proliferate in a self-organizing manner (Hernandez
and Palmer, 1988). It is thus plausible that the cia margins
themselves trigger fractionation in Passiflora.
The functional interaction between space and morphogenesis
is not fully understood. Based on mechanical models
(Prusinkiewicz and Barbier de Reuille, 2010) and molecular
findings (Hamant et al., 2008), mechanical pressure increases
cell wall stress and affects morphogenesis via the microtubule
cytoskeleton system. Braam (2005) reports that touch-induced
gene expression is widespread in Arabidopsis thaliana, indicat-
ing signal transfer between physical force and molecular pro-
cesses. The newly generated space in Passiflora can be
understood as negative mechanical pressure increasing mitotic
activity and/or loosening cell walls. It is assumed that meristem
widening changes cellular structures and stimulates molecular
processes in a similar way to pressure.
Homology of the corona
Homology of the corona in Passiflora has been controver-
sially discussed for almost 200 years. Thereby, anatomical
(Puri, 1947, 1948), developmental (Masters, 1871; Bernhard,
1999), systematic (Endress, 1996) and developmental genetic
(Hemingway et al., 2011) arguments were used to explain its
origin. All the data are worth considering, but only reflect a
single aspect of floral complexity.
Puri (1948) followed Lindley (1830) in assuming that the co-
rona originates from sepals and petals. His main argument was
the inverted course of the vascular bundles in some corona ele-
ments. de Wilde (1974) modified this view in interpreting the
corona elements as outgrowths of the receptacle being anatomi-
cally influenced by sepals and petals. However, while vascular-
ization was used to corroborate homologies in the past, the
belief in vascular conservatism is outdated today (Carlquist,
1969; Schmid, 1972; Bernhard, 1999). According to molecular
findings and computational modelling, the development of vas-
cular bundles depends on local auxin accumulation (Reinhardt
et al., 2003) and tissue growth (Runions et al., 2005; summa-
rized by Prusinkiewicz and Runions, 2012). Thus, especially in
late developing structures, the orientation of bundles should not
be overinterpreted (Puri and Agarwal, 1976). As regards the li-
men, Puri (1948) assumed a staminode origin as he noticed that
the limen has five tips in some species alternating with the an-
thers. Bernhard (1999) illustrated such a limen in P. racemosa;
Page 13 of 14
however, in the species investigated here, the limen remains a
low fold. It develops late in flower ontogeny as a ring-shaped
effiguration of the receptacle and does not show any pentamer-
ous pattern. Developmentally, there is thus no evidence for a
staminode origin. Given that the gaps between the young sta-
mens provide most space for limen development, the apparent
five tips may be the mere outcome of spatial constraints.
From a systematic point of view, Endress (1996) argued that
the corona elements may correspond to a group of modified
staminodes. He referred to the closely related family
Flacourtiaceae and compared the corona in Passiflora with a
polyandrous androecium. However, the present study confirms
the view of Bernhard (1999) that the genesis of the corona has
Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
almost nothing in common with the development of staminodes
or androecia. Corona elements arise much later than the floral
organs, appear in irregular numbers without any clear phyllo-
tactic pattern and initiate in some species in a convergent se-
quence not known from polyandric androecia (Ronse de Craene
and Smets, 1992; Ronse de Craene, 2010).
Nevertheless, developmental genetic studies in P. caerulea
appear to support the hypothesis of a staminal origin of the co-
rona. Hemingway et al. (2011) found B- and C-class gene ex-
pression in all corona elements and the androgynophore of this
species and B-class genes in its petals and limen. According to
the ABCDE model of floral evolution (Theißen, 2001), B-class
gene expression characterizes petals, and B- and C-class gene
expression characterizes stamens. Hemingway et al. (2011) pre-
liminarily conclude that the corona might be homologous to
stamens while the limen could be a novel structure. This argu-
ment raises the question of why gene expression characterizes
organ identity in the corona elements but not in the limen. If B-
class gene expression in the limen is compatible with its inter-
pretation as a novel structure, why should B- and C-class gene
expression not indicate structures ‘sui generis’ in the corona
area? Thus, as in Narcissus bulbocodium (Waters et al., 2013),
gene expression patterns do not easily explain the homology of
the corona.
In the present study, the corona in Passiflora was found to be
a novel structure not derived evolutionarily from petals or sta-
mens. Instead, the ancestral flower might have had the capacity
to expand during development. Dependent on position and rela-
tive timing of expansion and fractionation, different lineages
could have evolved. If stamen primordia were affected by ex-
pansion, polyandrous flowers would have originated, and if the
space between floral organs was widened, additional structures
would have appeared. Thus, there is no need to homologize the
corona with staminodes, even if related families have a polyan-
drous androecium. As to the expression of B- and C-class genes
in the corona, homology with stamens is also not imperative.
Proceeding from a centripetal gradient of floral identity gene ex-
pression, B- and C-class genes might be already activated in the
hypanthial zone before the corona arises. However, why C-class
genes are not expressed in the limen is an unresolved question.
CONCLUSIONS
Passiflora flowers illustrate exciting dynamics during develop-
ment. After the fractionation of floral organ primordia, interca-
lary mitotic activity elevates sepals and petals by a hypanthial
Page 14 of 14 Claßen-Bockhoff and Meyer — Corona formation in Passiflora
cup which itself widens and gives rise to the corona. The andro-
gynophore is the outcome of local meristem activity as well as
the limen. Thus, all peculiar flower structures are closely corre-
lated with additional growth processes in the developing
flower.
Although the developmental dynamics in flowers are well
known, the reference system for flower evolution is rather
static: a shoot tip of limited growth producing floral leaves and
organs in a determinate sequence. Differences from the proto-
type are explained by heterotopy and heterochrony
(Hemingway et al., 2013) without considering time and space
as factors on their own, inherent to the system. As long as adult
structures are compared, the need for homologizing corona ele-
ments with floral organs is evident. However, if the relative
timing of element formation during development plays a role,
corona elements and anthers are not homologues, and, if the
generation of new space has a meaning, the appearance of
novel structures should be expected. The study of corona for-
mation in Passiflora thus supports a more dynamic concept of
the flower (Claßen-Bockhoff, 2015), integrating molecular,
phylogenetical and morphogenetic data.
ACKNOWLEDGEMENTS
We thank M. Drews, B. and T. Ulmer, Witten, for providing
us with plant material, and H. Bahmann for cultivating the
plants in the Botanical Garden at Mainz University. We also
gratefully acknowledge stimulating discussion with
P. Prusinkiewicz and A. Runions (both Calgary, Canada) and
helpful comments by L. Ronse De Craene (Edinburgh) and a
second reviewer.
REFERENCES
Abizza LC. 2010. Desenvolvimento da corona em flores do gênero
Passiflora (Passifloraceae). PhD thesis, Universidade Estadual de
Campinas.
Arber A. 1937. Studies in flower structure. III. On the corona and androecium in
certain Amaryllidaceae. Annals of Botany 1: 293–303.
Bernhard A. 1999. Flower structure, development, and systematics in
Passifloraceae and Abatia (Flacourtiaceae). International Journal of Plant
Science 160: 135–150.
Braam J. 2005. In touch: plant responses to mechanical stimuli. New Phytologist
165: 373–389
Carlquist S. 1969. Toward acceptable evolutionary interpretations of floral anat-
omy. Phytomorphology 19: 332–362.
Čelakovský LJ. 1898. Ueber die Bedeutung und den Ursprung der Paracorolle
der Narcisseen. Bulletin international de l’Academie des sciences de
Prague 5: 1–15.
Chen S. 1971. Developmental morphology of the flower in Narcissus. Annals of
Botany 35: 881–890.
Claßen-Bockhoff R. 2015. The shoot concept of the flower: still up to date?
Flora (in press)
Claßen-Bockhoff R, Bull-Hereñu K. 2013. Towards an ontogenetic under-
standing of inflorescence diversity. Annals of Botany 112: 1523–1542.
Eichler AW. 1875. Blüthendiagramme construirt und erläutert. 1. Leipzig:
Theil. (repr. Eppenheim: Koeltz 1954).
Endress PK. 1996. Diversity and evolutionary biology of tropical flowers.
Cambridge: Cambridge University Press.
Glück H. 1919. Blatt- und blütenmorphologische Studien. Jena: Fischer.
Goebel K. 1933. Organographie der Pflanzen insbesondere der Archegoniaten
und Samenpflanzen, 3rd edn. Teil. Jena: Fischer.
Hamant O, Heisler MG, Jönsson H, et al. 2008. Developmental patterning by
mechanical signals in Arabidopsis. Science 322: 1650–1655
Hemingway CA, Christensen AR, Malcomber ST. 2011. B- and C-class
gene expression during corona development of the blue passionflower
(Passiflora caerulea, Passifloraceae). American Journal of Botany 98:
923–934.
Hernandez LF, Palmer JH. 1988. Regeneration of the sunflower capitulum af-
ter cylindrical wounding of the receptacle. American Journal of Botany 75:
1253–1261.
Janzen DH. 1968. Reproductive behavior in the Passifloraceae and some of its
pollinators in central America. Behavior 32: 33–48.
Krosnick SE, Freudenstein JV. 2005. Monophyly and floral character homol-
ogy of old world Passiflora (Subgenus Decaloba: Supersection Disemma).
Systematic Botany 30: 139–152.
Krosnick SE, Harris EM, Freudenstein JV. 2006. Patterns of anomalous floral
development in the Asian Passiflora (Subgenus Decaloba): supersection
Disemma). American Journal of Botany 93: 620–636.
Kugler EE, King LA. 2004. A brief history of the passionflower. In: T Ulmer,
Downloaded from http://aob.oxfordjournals.org/ at University of California, San Diego on December 17, 2015
JM MacDougal, B Ulmer, eds. Passiflora – passionflower of the world.
Portland, OR: Timber Press, 15–26.
Leins P, Erbar C. 2010. Flower and fruit. Morphology, ontogeny, phylogeny,
function and ecology. Stuttgart: Schweizerbart.
Lindley J. 1830. An introduction to the natural system of botany. London:
Longman.
Masters MT. 1871. XIX.Contributions to the natural history of Passifloraceae.
Transactions of the Linnean Society of London 27: 593–645.
Meerow AW, Chase MW, Fay MF, Guy CL, Li Q, Zaman FQ. 1999.
Systematics of Amaryllidaceae based on cladistic analysis of plastid RBCL and
TRNL-F sequence data. American Journal of Botany 86: 1325–1345.
Moncur MW. 1988. Floral development of tropical and subtropical fruit and
nut species. Colllingwood, Australia: Commonwealth Scientific and
Industrial Research Organization.
Prusinkiewicz P, Runions A. 2012. Computational models of plant develop-
ment and form. New Phytologist 193: 549–569.
Puri V. 1947. Studies in floral anatomy IV. Vascular anatomy of the flower
of certain species of the Passifloraceae. Botanical Society of America 34:
562–573.
Puri V. 1948. Studies in floral anatomy V. On the structure and nature of the co-
rona in certain species of the Passifloraceae. Journal of the Indian Botanical
Society 27: 130–149.
Puri V, Agarwal RM. 1976. On accessory floral organs. Journal of the Indian
Botanical Society 55: 95–114.
Prusinkiewicz P, Barbier de Reuille P. 2010. Constraints of space in plant
development. Journal of Experimental Botany 61: 2117–2129.
Reinhardt D, Pesce ER, Stieger P, et al. 2003. Regulation of phyllotaxis by
polar auxin transport. Nature 426: 255–260.
Ronse De Craene LP. 2010. Floral diagrams. An aid to understanding flower mor-
phology and evolution. Cambridge: Cambridge University Press.
Ronse De Craene LP, Smets EF. 1992. Complex polyandry in the Magnoliatae,
definition, distribution and systematic value. Nordic Journal of Botany 12:
621–649.
Runions A, Fuhrer M, Lane B, Federl P, Rolland-Lagan AG, Prusinkiewicz,
P. 2005. Modeling and visualization of leaf venation patterns. ACM
Transactions on Graphics 24: 702711.
Runions A, Smith RS, Prusinkiewicz P. 2014. Computational models of auxin-
driven development. In: E Zazı́malova, J Petrašek, E Benkova, eds. Auxin
and its role in plant development. Heidelberg: Springer, 315–357.
Schaeppi H. 1939. Vergleichend-morphologische Untersuchungen an den
Staubblättern der Monokotyledonen. Nova Acta Leopoldina (N.F.) 6: 389–447.
Schmid R. 1972. Floral bundle fusion and vascular conservatism. Taxon 21:
429–446.
Singh V. 1972. Floral morphology of the Amaryllidaceae. I. Subfamily
Amaryllidioideae. Canadian Journal of Botany 50: 1555–1565.
Theissen G. 2001. Development of floral organ identity: stories from the MADS
house. Current Opinion in Plant Biology 4: 75–85.
Troll W. 1938–39. Vergleichende Morphologie der höheren Pflanzen. 1, Band 2.
Teil. Berlin: Borntraeger.
Velenovský J. 1910. Vergleichende Morphologie der Pflanzen. 3. Teil: Die
Morphologie der Blüte der Phanerogamen. Prag.
Waters MT, Tiley AMM, Kramer EM, Meerow AW, Langdale JA, Scotland
RW. 2013. The corona of the daffodil Narcissus bulbocodium shares
stamen-like identity and is distinct from the orthodox floral whorls.
The Plant Journal 74: 615–625.
de Wilde WJ. 1974. The genera of tribe Passifloreae (Passifloraceae) with
special reference to flower morphology. Blumea 22: 37–50.