Plant Syst. Evol.
249: 173–189 (2004)
DOI 10.1007/s00606-004-0203-6
Contributions to the functional anatomy and biology
of Nelumbo nucifera (Nelumbonaceae)
III. An ecological reappraisal of floral organs
S. Vogel and F. Hadacek
Institute of Botany, University of Vienna, Vienna, Austria
Received November 6, 2003; accepted June 1, 2004
Published online: October 18, 2004

Springer-Verlag 2004
Abstract. The flower of Nelumbo nucifera dis-
plays some highly specialized structural details
apparently related to its pollination ecology.
The functional significance of these structures,
however, is still under debate. This paper
presents new anatomical and chemical data
that contribute to our knowledge of the floral
life of the lotus. Special focus is directed on the
source of scent and heat production, the role of
the staminal appendages, and stigma charac-
teristics. The receptacle, the staminal append-
ages and connectives are covered with a
papillose, osmogenous epithelium and func-
tion as osmophores. A fractioned GC-analysis
revealed that mainly 1,4-dimethoxybenzene
(1,4-DMB) is emitted by these parts, whereas
high amounts of C15 – C17-alkanes, n-alkenes,
n-alcadienes and n-alkatrienes dominated the
volatile blend that was diffusely emitted by the
petals. In accordance with the literature,
absolute predominance of 1,4-DMB and pen-
tadecane was found, while some differences
occurred in minor components. The active
source of heat generation, apparently a means
to enhance fragrance volatilization, was the
receptacle. The compact mesenchyma under-
lying its upper platform and flanks is glandular
and rests on a storage zone replete with starch
that becomes exhausted during the metabolic
flare-up. Spot measurements confirm the
flower’s capacity to maintain a temperature
level of ca. 31 C by compensating the envi-
ronmental temperature fluctuations. The sta-
minal appendages warm up because they are
close to the receptacle; they are not actively
thermogenous, as formerly proposed. Their
starch load remains nearly unaffected until
defloration. The appendages, besides contrib-
uting to olfaction, may represent food bodies.
Along with the pollen – shed at the male stage
of anthesis – the appendages may function as a
primary attractant for coleopteran pollinators,
an interpretation corroborated by the remain-
ing floral traits, which suggest the cantharoph-
ilous syndrome. Published field data, so far
only available for the American subspecies,
reveal variable mixed assemblages of insect
visitors; no feeding on the staminal append-
ages is recorded. Possibly, the system once
involved a partnership that has not persisted
during the long history of the genus, which is
nowadays attended by opportunists.
Key words: Cantharophily, floral ecology, floral
receptacle, floral scent, food body, osmophore,
protogyny, thermogenesis.
174 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo

Introduction and general traits of the flower carpels are separately and distantly embedded
in a receptacle, which is an axial, fleshy
The flower of the sacred lotus, Nelumbo
emergence shaped like an inverted, truncate
nucifera, exhibits a multitude of interesting
cone. The receptacle’s upper, flat platform
aspects and has been the subject of numerous
(3 – 4 cm in diameter) is perforated by
investigations. Detailed descriptions of the
circular pits, allowing each of the button-like
flower were provided, among others, by
stigmata to become exposed on a very short
Schneider and Moore (1977), Sohmer and
style. After anthesis, the receptacle enlarges
Sefton (1978), Schneider and Buchanan
and becomes a dry, spongy, caducous carpo-
(1980), Moseley and Uhl (1985), Williamson
phore able to float, bearing nutlets of extreme
and Schneider (1993) and Borsch and Barth-
longevity.
lott (1994). Ontogeny was studied by Hayes et
Anthesis lasts 2(3) days and undergoes
al. (2000), anther and ovule morphology by
drastic changes of flower shape (Figs. 1A, 2 A).
Gupta and Ahluwalia (1977) and Igersheim
On the first day (D-1), when the blossom opens
and Endress (1998), embryology by Ito (1986)
around 7:00, the petals unfold only with their
and vascularization by Khanna (1965).
distal halves; these form a bowl with an
Several authors have focussed on the ecol-
opening 2 – 5 cm across, which widens towards
ogy and physiology of the floral life of
noon. With unfolding, the interior production
Nelumbo. As some of their conclusions are
of scent and also of warmth commences.
controversial, we attempted to clarify some
Around 16:00, the perianth recloses to almost
disputed points based on our own observations
bud shape and remains so until ca. 7:00 of the
made on N. nucifera in the Botanical Gardens
second day (D-2), while scent and warmth
of Mainz and Vienna. These concern: (1) The
production continues. Then it opens again, but
source of scent production, (2) The source of
now completely, achieving a shallow, rotun-
heat generation, (3) The role of the staminal
date disk-shape
20 cm in diameter (Figs. 1B,
appendages, (4) The characteristics of the
2B). In the afternoon of D-2 , the perianth
stigma. In a separate chapter, the pollination
closes once more, but less tightly, and starts to
ecology will then be discussed in the light of
wither; on the third morning it expands again,
the floral features investigated and the avail-
but in the course of D-3 the perianth and
able field data.
stamens abscise.
The large, solitary blossoms of Nelumbo
The flowers are protogynous. Stigmata are
nucifera rise up to 1 m above the water
receptive through D-1, while the androecium
surface on a pedicel axillary to a bract
remains enclosed; the androecium is tightly
preceding a foliage leaf. They are polymerous
appressed to the receptacle side walls by the
throughout. The 18–28 spirally inserted peri-
still unexpanded basal halves of the petals,
anth segments are differentiated without a
allowing only the sterile appendages to emerge
clear-cut boundary into a few smaller pale-
and become exposed (Fig. 2A). The flower is
green sepals (following Hayes et al. [2000] the
thus strictly herkogamous at D-1. With the
two lowermost only are true sepals) and rose-
complete expansion on D-2, the flower enters
coloured, UV- absorbing, ovate petals of
its male stage. The now exposed stamens
distally increasing size. The more than 200
spread, and the anthers dehisce; much of the
stamina consist of an initially very short (9–
yellow pollen falls down, collecting at the
7 mm) filament, the linear, much elongated
perianth’s floor (Fig. 2B). Receptivity contin-
(ca. 2 cm), latrorse/introrse, yellow anther,
ues, however, at least during D-2. The stigmata
and a terminal, connectival, cylindrical, stipi-
are selfcompatible (Schneider and Buchanan
tate, ivory-white appendage 3.5–5 mm long.
1980), but autogamy, unless insects mediate it,
The gynoecium is superior and apocarpous,
remains excluded.
with usually 15 – 20 uniovulate carpels; the
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo 175

Fig. 1. Floral movements of Nelumbo nucifera during anthesis, schematically. A Flower on D-1 (female stage),
B Flower on D-2 (female and male stage). a position of petals at noon of D-1, b position of petals during the
first and second night, s stigma, r receptacle, an anther (in B dehisced), ap staminal appendage

Material and methods
Flowers of Nelumbo nucifera subsp. nucifera were
taken from plants cultivated in a greenhouse of the
Botanical Garden of Mainz (Germany) and in an
open-air basin of the Botanical Garden of Vienna
(details under Material and Methods in part I of
this series [Vogel 2004]). Vital staining of the
osmophores was performed using neutral-red in a
1: 10 000 watery solution (pH ¼ 7), applied to
hand-cuttings. For other observations of fresh
material, a freezing microtome was used.
Analysis of scent components: Whole fila-
ments, anthers, antheral appendages, as well as
the distal and proximal half of one petal were
collected on D-1 of anthesis on 9 July 2003 from a
flowering colony cultivated in the Botanical Gar-
den of the University of Vienna. These parts were
placed into headspace vials (Perkin Elmer
B104235), and sealed with PTFE-coated rubber
septa (Perkin Elmer B104239). The whole recepta-
cle was transferred into a 250 mL beaker that was
sealed with parafilm. Odour collection was per-
formed by applying the solid phase microextraction
methodology (SPME) with a 65 lm poly-
dimethylsiloxane/divinylbenzene fibre (Supelco
57310-U) in a manual fibre holder (Supelco
57348-U). Volatile collection in the headspace of
each sample lasted for 30 min. GC–MS analyses
were performed on a Turbomass quadrupole mass
spectrometer (Perkin Elmer), directly linked to an
Autosystem gas chromatograph (Perkin Elmer).
The fibre holder was inserted into the split-splitless
injector port at 250 C. The liner used was a special
liner for SPME sample injections (0.75 mm ID,
Perkin Elmer 2631201). All injections were carried
out in the splitless mode.The column used was a
polar PE-WAX (60 m, 0.25 mm ID, 0.25 lm
polyethylene glycol film, Perkin Elmer N931-
6013). The CG oven was programmed to stay
initially at 40 C for 3 min, to increase from 40 to
120 C at a rate of 6 C/min)1, from 120 to 180 C
at a rate of 10 C/min)1 and finally to stay at
220 C for 21 min. Helium was used as carrier gas
at a flow rate of 1 mL min)1. The transfer line of
the MS was thermostatically controlled at 250 C.
The temperature of the ion source was 150 C.
Mass spectra were recorded from 40 – 400 amu per
0.6 sec in the electron ionisation mode (EI) at an
electron energy of 70 eV and a filament emission of
200 lA. Tentative identification of the odour
compounds was carried out by careful comparison
with commercially available mass spectra libraries,
Wiley Registry of Mass Spectral data, 6th edition,
NIST/EPA/NIH Mass Spectral Library 1.5a, and
ADAMS (2001). TIC signal peaks were integrated
to express relative amounts of volatiles as the
percentage of the total peak area.
Temperature readings on the flower were taken
with a thermocouple connected with a Fluke 52
digital thermometer (resolution 0.1 C). Permanent
slides for LM were prepared by the paraffin
method, cut with a Reichert-Jung rotary micro-
tome 2050 (7 lm), stained with hematoxylin/safra-
nin and mounted in Euparal.
176 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo

Fig. 2. Floral stages, receptacle and staminal appendages of Nelumbo nucifera. A Receptacle and staminal
appendages on D-1 at noon. B Flower on D-2. C Longisection of receptacle: part of platform and edge. D Part
of platform and subjacent glandular tissue, enlarged. E, F Staminal appendages in transection, in E on D-1, in F
at the end of D-2 with starch contents largely conserved. e epithelium, st stoma, h tanniferous hypodermis, g
glandular mesenchyma
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
Results
Scent production
The flowers develop a powerful scent. Scent
emission starts with the first opening and
continues throughout anthesis. The perfume
was characterized as benzene-like, heavy,
sweetish, syrup-like, ‘‘chemical’’, or ‘‘medici-
nal’’ by a number of test persons. It is strong
enough that, as Robertson (1889) and Schnei-
der and Buchanan (1980) attest, honey bees
and other insects died after several hours when
enclosed in D-1 flowers.
Scent emission is apparently not diffuse but
confined to certain parts of the flower. Schnei-
der and Buchanan (1980) concluded that
stamens, pollen and stigmata were chief
sources, while Sohmer and Sefton (1978) stated
that the top of the receptacle might be
responsible. Our own olfactory tests of the
perfume of different floral organs that were
enclosed in glass vials also suggested that the
receptacle plays a major role in scent genera-
tion, in congruence with Sohmer and Sefton’s
(l.c.) observations.
Gas-chromatography of the volatiles has
already been carried out by two research
groups. Mookherjee et al. (1990) analyzed the
odour of the whole flower and reported the
presence of 6 compounds in the headspace.
Omata et al. (1991) and Nakanishi and
Yomogita (1999) concentrated on stamens
and petals and found 32 compounds in the
headspace and about 70 compounds in the
n-pentane extracts. To obtain an even more
differentiated spectrum of volatile emission
within the lotus blossom, we separately deter-
mined the volatile compounds that were pres-
ent in the headspace of the receptacle, the
anthers, the staminal appendages, and the
proximal and distal halves of the petal.
Differential GC analysis of scent
components
We tentatively identified 35 compounds in all
analysed headspace samples. The results
177
showed that all flower parts were involved in
scent emission, but there was considerable
variation in quality and quantity of the emitted
compounds in the different floral parts of the
investigated plant. The detected compounds
comprised three broad classes of volatiles:
aliphatic hydrocarbons, aromatic compounds
and terpenoids. Table 1 summarizes the
results.
The receptacle, the anthers and the stami-
nal appendages emitted 1,4-dimethoxybenzene
(1,4-DMB) in a relative amount exceeding
90% of total emitted volatiles. The monoter-
pene a–terpinene occurred as a minor com-
pound in the anthers (5.4%), the monoterpene
aldehyde b–cyclocitral in the receptacle
(4.2%); cis-jasmone and the sesquiterpene a–
farnesene were near-trace compounds in both
organs. The appendages additionally emitted
the sesquiterpenes farnesal and methyl farne-
senoate. The irregular monoterpene ionol was
only detected in the receptacle.
The proximal and distal half of the petal
differed considerably from the former organs,
both quantitatively and qualitatively. Alt-
hough our methodology did not allow absolute
quantification of volatiles, the results suggest
that, considering all petals together, by far the
most emitted volatiles originate from the distal
half of the petals. The predominating com-
pounds in the material we investigated
consisted of C15–C17 aliphatic hydrocarbons.
The major components were pentadecane,
heptadecene isomers and heptadecadiene.
1,4-DMB was present only as a minor com-
pound (3.3%). The monoterpene cis-sabinene
hydrate was the only representative (in a near-
trace amount: 0.3 %) of that class of com-
pounds, which usually dominates volatile
blends.
In accordance with previously published
analyses, the most prominent compounds
were 1,4-DMB and a series of saturated and
unsaturated C 15 and C 17 hydrocarbons.
The latter were emitted by the petals as
dominating volatiles. Especially the distal part
emitted most of the hydrocarbons along with
178 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo

Table 1. Identified floral volatiles in the headspace of the receptacle, the anthers, anther appendages, and
basal and distal parts of the petals, analysed by SPME-GC-MS (for experimental details see Materials and
Methods). tr = traces
Compounds Receptacle Anthers Anther append. Petal basal Petal distal
Aliphatic carbohydrates
n-alkanes
Undecane 2.1 0.1
Tetradecane 4.8 0.2
Pentadecane1 0.1 45.0 56.0
Hexadecane 0.2
Heptadecane1 1.1 1.4
Octadecane 0.1
n-alkenes
Pentadecene1,2 0.1 16.5 7.8
Hexadecene2 0.2
Hexadecene isomer1,2 0.1
Hexadecene isomer2 0.1
Hexadecadiene isomer1,2 0.1
Hexadecadiene isomer2 tr
Heptadecene isomer1,2 4.5 8.0
Heptadecene isomer2 3.6 6.9
Hexadecadiene2 tr
Heptadecadiene isomer1,2 4.1 13.8
Heptadecadiene isomer2 0.1
Heptadecadiene isomer2 0.1
Heptadecatriene2 1.4
Octadecene 0.1
Ketones
Cis-jasmone1 0.3 0.7
Alcohols
Isopentyl alcohol 3.3
Aromatic compounds
1,4-dimethoxybenzene1 93.7 90.4 99.3 4.7 3.3
Terpenoids
Monoterpenes
Sabinene1 4.5
b-myrcene1 1.9
a-terpinene1 5.4 1.9
Limonene1 1.7
c-terpinene1 3.2
1,8-cineole1 3.4
b-cyclocitral 4.2
Cis-sabinene hydrate 1.0 0.3
Sesquiterpenes
a-farnesene 0.5 0.2
Farnesal 0.2
Methyl farnesenoate 0.3
Irregular
Ionol 1.3
1
Compounds that have already been reported by previous investigators.
2
Unambiguous determination of position of double bonds was impossible based on the available MS spectra.
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
1,4-DMB, which was the main component in
the anthers, their appendages and the recep-
tacle. In view of the amounts of tissue
analyzed the distal part of the petal produced
by far the most volatiles emitted in all
analyzed samples. Among those, heptadecane,
n-heptadecene and n-heptadecadiene isomers
were the most prominent. The otherwise
typical floral monoterpenes occurred only in
the proximal half of these phyllomes. Small
quantities of cis-jasmone and the sesquiter-
pene a-farnesene were confined to the three
non-petal organs. Of the minor components,
b-cyclo-citral and ionol only appeared in the
volatile blend of the receptacle, the monoter-
pene a-terpinene and isopentyl alcohol only in
the anthers, and the sesquiterpenes fanesal
and methyl-farnesenoate only in the append-
ages.
Comparing the sums of all integrated
peaks, the proximal part of the petal emitted
40 times lower amounts of volatiles than the
distal part. In contrast to the distal part, a
number of monoterpenes comprised only
about 18% of the relative total amount of
emitted volatiles of that tissue. Of the hydro-
carbons characteristic for the volatile blend of
the proximal part, only the major components
were found. The relative amounts of 1,4-DMB
resembled the distal part, but the actual peak
area amounted to only 3% of that of the distal
part.
Our results support earlier studies, but
disagree in some aspects: (1) Omata et al.
(1991) published a chromatogram of a head-
space analysis of the stamens that showed
abundant pentadecane and pentadecene. In
our samples, those two compounds appeared
only as trace compounds in the appendages.
Omata et al. investigated many cultivars of
lotus and found that the ratio of 1,4-DMB and
pentadecane was quite variable in the head-
space. Our results fall within that variation. 2)
Omata et al. (1991) further reported several
monoterpenes in the headspace of the fila-
ments that our sample lacked. In contrast, we
detected most of those monoterpenes in the
proximal half of the petal. Based on our
179
current knowledge, these discrepancies cannot
be explained, but might reflect variability
between the cultivars. (3) Omata et al. (1991)
report the monoterpenes terpinolene, linalool,
terpineol and terpinen-4-ol as well as the
sesquiterpene caryophyllene to be prominent
constituents of the volatile blend of the
stamens. We failed to detect these volatiles in
any of our samples, but instead found
a-farnesene in the receptacle and in the stami-
nal appendages, as well as farnesal and methyl
farnesenoate in the staminal appendages.
Omata et al. (1991) report only pentadecane
as a major compound of the petals. Unfortu-
nately, the authors present only a headspace
analysis for the stamens alone. However, the
dominating compounds in their solvent extract
analysis comprise higher-weight n-alkanes that
are usually specific components of epicuticular
waxes. The scope for the interpretation about
possible merits in attracting pollinators
remains limited.
Anatomical aspects of odour production
Receptacle. The epidermis of this axial struc-
ture is a glandular epithelium. This layer
covers the yellowish upper platform in which
the carpels are embedded, and also lines the
upper two thirds of the side walls. The
epithelium consists of columnar, papillose cells
with dense cytoplasmic content (Fig. 2C, D),
on the platform with numerous sunken sto-
mata. The epidermis is separated (like most
other floral parts) from the mesenchyma by a
tanniferous hypodermis. The subjacent tissue
is again of glandular appearance; it is a
compact yellowish body ca. 18–20 layers thick
(0.6 mm) on the top and ca .12 layers thick at
the flanks (0.4 mm). It lacks interstices, is well
supplied with open vascular bundles, and is
interspersed with single tanninic and crystal
idioblasts. The glandular cells are moderately
cytoplasmic and in D-1 flowers the proximal
layers contain starch. The non-glandular
parenchyma below, although spongy due to a
system of large air caverns, contains the main
storage of starch at D-1 stage. The starch
180 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
content becomes largely exhausted at the end
of D-2, except for that contained in the
stomata.
Active osmogenous tissues can be detected
by selective vital staining with neutral red
(Vogel 1963, 1990). Unfortunately, the method
is often applied uncritically. Schneider and
Buchanan (1980) reported red dying of the
stigmata, the anthers and pollen. They con-
cluded, as mentioned above, that these parts
should be osmogenous. This reaction is, how-
ever, insufficient to deduce scent generation if
the colour hue and anatomical circumstances
are not considered. Neutral red also tinges
non-glandular tissue including anthers, pollen,
stigmata and necrotic cells; the resulting colour
is orange to brick-red. In contrast, the hue of
diagnostic value, achieved by neutral red
solution (pH
7) in active glands is a deep
bluish or raspberry red. This is caused by rapid
accumulation of pigment molecules in the
vacuole, where they become chemically bound
to temporarily present by-products involved in
the secretion process. By this reaction, a sink is
maintained for further permeation of pigment
molecules (Höfler 1954). The scent substances
themselves, which originate in the cytoplasm,
are not directly tinged. This mechanism also
works in other gland types such as nectaries.
We applied the neutral red method with the
above-mentioned precautions, using fresh
microscopical slices of the receptacle, and
obtained a strong raspberry red vacuole tinc-
tion in the glandular epidermal and the sub-
jacent glandular tissue. Schneider and
Buchanan (l.c.) failed to stain the receptacle
tissue because they possibly administered neu-
tral red solution to the entire receptacle en
bloc; this would have led stigmata react readily
(but unspecifically), while the pigment was
unable to permeate the rather thick and
unwettable axial epidermal walls.
The complex thus exhibits a stratification
characteristic of many scent glands: emission
layer (epidermis), production tissue and stor-
age tissue. The first author (S.V.) therefore
postulated, in a preliminary communication
(1993), that the receptacle represented a typical
osmophore, in analogy to those of aroids, etc.
The new GC results reveal the participation of
other floral parts, requiring modification of
this opinion. Based on the features of the
staminal appendages and on heat production
(see below), the epidermal epithelium alone
appears to be osmogenous.
Staminal appendages. These cylindrical
bodies have a large surface and probably
contribute more to scent production than the
receptacle. 1,4-DMB is absolutely dominant in
both organs (Table 1). The bodies of the
staminal appendages are covered throughout
with a glandular epithelium whose densely
cytoplasmic cells are less columnar, but elon-
gate papillose and thin-walled. Stomata are
absent here; positive neutral red reaction is
confined to this layer, and the tanninic hypo-
dermis is present. The remaining mesophyll,
with a single central bundle, consists of storage
parenchyma with small interspersed intercellu-
lar gaps and single tanniferous cells but no
calcium oxalate druses. Appendages taken
from D-2 flowers at 17:00 were somewhat
shrunk, the epithelium necrotic and the inters-
titia enlarged to radial stripes at the expense of
storage cell size. Some radial sectors showed
empty cells; most starch, however, was pre-
served (Fig. 2E, F). The presence of starch was
proved by applying iodine solution to hand-
cuttings cleared with chloral hydrate and by
comparison with cuttings of D-1 appendages.
The result argues against the involvement of
the mesophyll in scent production, which thus
is confined to the epidermis, as in the recep-
tacle. Nevertheless, the bodies can be termed
osmophores and the starch load serves some
other function.
Anthers. These are also considerably
scented, even though they are hidden under
the petal bases at D-1. Small amounts of
a-terpinene and isoamylalcohol, which are
confined to the anthers, may originate from
the pollen, but 1,4-DMB is probably produced
in the papillose epithelium that lines the
adaxial connective surface.
Petals. They bear a papillose epidermis on
their inner flank, but this epidermis is not
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
glandular. The scent emanation of the petals
mainly contains hydrocarbons and appears to
be diffuse, as is common in most scented
flowers. A smelling test revealed that only the
inner flank is scented.
Heat production
Thermogenicity, the most remarkable feature
of the flower, was discovered by Miyake (1898)
in the Asian lotus. He noted a temperature rise
by as much as 10 C above ambient. The
source of the heat was not specified. Schneider
and Buchanan (1980) extended this finding to
the American subspecies, measuring here an
excess of up to 6 C. Recently, thermogenicity
in Asian Nelumbo was the subject of extensive
physiological research by Seymour and
Schultze-Motel (1996, 1998), Seymour et al.
(1998) and Lamprecht et al. (1998). The
unexpected outcome was that the flower has
the capacity to rather precisely regulate heat
generation. It manages to maintain 30 – 35 C
during anthesis against fluctuations of ambient
temperatures ranging from 10–30 C. This
resulted in differences of 10 C on cool nights
and differences approaching zero during the
daytime in sunny weather. This was accompa-
nied by variation in oxygen consumption. A
similar homoeothermic regulation was previ-
ously established only in araceous plants
(Philodendron, Nagy et al. 1972, Seymour
et al. 1983, and Symplocarpus, Knutson
1974). This phenomenon clearly has adaptive
significance.
The question about which parts generate
heat has been evaluated differently by various
workers. Comi (1939) claimed the staminal
appendages as the site of thermogenesis. Indi-
rectly based on Comi’s results, Skubatz et al.
(1990), working on the American lotus, found
similarities in metabolism and ultrastructure
with the thermo-(and osmo-) genic spadix
tissue of the arum lily. This corroborated the
interpretation that the appendages produce the
heat.
Neither Comi’s nor Skubatz et al.’s con-
clusions were drawn from direct temperature
181
measurements of the appendage, but from
circumstantial features. Comi had noted the
high starch content and determined that this
material diminished in aged flowers. The
analogy with the aroid spadix, with its rapid
respiratory combustion of starch reserves in
the process of heating-up (and odour produc-
tion), prompted her to ascribe that role to the
appendages of Nelumbo. This interpretation
was supported by Skubatz et al. (1990), who
recognized an elevated number of mitochon-
dria in the appendix cells. The authors also
reported the occurrence of the ‘uncoupled’,
cyanide-resistent respiration pathway in these
bodies, as is typical for the mentioned araceous
inflorescences.
In order to examine whether the staminal
appendages themselves actively produce
warmth, I removed appendages from a D-1
flower around noon, packed them immediately
into a tightly fitting cavity made in styrofoam,
and measured the temperature against a dis-
tant portion of the styrofoam bed. Reading
was done after slightly pressing the sensor
plate (4 mm Ø) of a thermocouple upon the
appendage mass or, alternatively, by piercing a
sensor needle 3–4 mm into the sample.
In no case did temperature rise beyond a
few tenths of a centigrade compared to the
surrounding material, and this difference
dropped within a few minutes to zero. Note
again the insignificant losses of reserve mate-
rial in D-2 appendages versus major losses in
the receptacle.
A possible explanation for the inconsis-
tency with previous records is that Comi’s
samples were brought to the laboratory from
an outside culture and fixed after the trans-
port, during which they may have been
negatively affected. The ultrastructural fea-
tures are hardly specific for thermogenous
cells, but occur in other glandular cell types
as well. The presence of a metabolism
otherwise found in thermogenous tissues
remains obscure in the absence of heat
production. However, the occurrence of such
a metabolism is no longer necessarily conclu-
sive, because it has now been detected in
182 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
other kinds of plant tissue (Lambers 1985). In
D-1 flowers, appendages and anthers appar-
ently warm up by conductivity because they
are so close to the receptacle. In D-2 flowers,
however, they spread in many directions.
Even if the appendages were actively thermo-
genous, the heat would continuously fade due
to the small size but large overall surface area
of these bodies. Considering the absence of
thermogenous tissue and the effect of neigh-
bour radiation, I question Seymour and
Schultze-Motel’s (1996) statement that ‘‘the
receptacle is responsible for about one half of
active heat generation, and petals and sta-
mens about a quarter each’’.
The receptacle as source of the heat
Initially unaware of previous records, one of us
(S.V.), felt the warmth by chance when touch-
ing the receptacle with his lip. Further
experimentation convinced him that the heat
originates in this organ (Vogel 1993). Com-
parative temperature measurements of this
organ were made between 1990 and 2003 in
greenhouse and outdoor cultures. Few flowers
were available per year, and usually only a
single flower could be observed at any one
time. Readings were manually taken around
Fig. 3. Heat production of the receptacle; surface
temperatures at noon of D-1 (example)
noon in shaded D-1 and D-2 flowers (N=22
and N=7, respectively) by placing the ther-
mocouple sensor plate tightly upon the sur-
face, or by piercing a sensor needle 2 – 3 mm
into the receptacle. Pierced samples were used
only once. Four points of the surface were
chosen (Fig. 3): centre (A) and rim (B) of the
upper surface, submarginal zone (C) and side
wall 15 mm below the edge (D). Ambient
temperature was measured concomitantly.
The warmth of the receptacle averaged
6.3 C above ambient temperature (range 12.1
– 0.1 C on both anthetic days. The mean
differences were 0.3 C (N=29) between A and
B, 0.24 C between A and C, and 0.75 C
(N=28) between A and D. These positive
differences were more marked in D-1 than in
D-2 flowers, no doubt because the flanks of the
receptacle were protected and isolated by the
appressed immature androecium during D-1
anthesis. Nevertheless, the average relative
excess was still 0.7 C at D-2 when the flanks
became exposed; this indicates that interior
conditions also play a role, i.e. that the heat is
mainly seated near the level of the storage
zone.
The extent to which the receptacle heated
up responded to the ambient temperature. This
organ maintained an average of 31.3 C
despite a range of +18.7  to +33.0 C
outside at daytime (Diagram 1). This agrees
with the compensative, homoeothermic regu-
lation capacity found by Seymour et al. (1998)
in the Nelumbo flower, which, remarkably, is
also operative at night. For example when
testing a D-1 flower at 23:30 and at an ambient
temperature of 22.8 C, we found an excess in
A of 10.4, in B of 9.8, in C of 10.8 and in D of
11.3 C. As experimentally established by the
above-mentioned authors, this regulation is an
autonomous response to ambient temperature
and is independent of light periodism; light
periodism does, however, trigger the perianth
movements.
The heat produced by the receptacle sec-
ondarily warms up the flower’s free interior as
well as the adjacent stamens and petals; this
clearly only occurs at the closed or semi-closed
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo 183

Diagram 1. Differences between temperature of the receptacle and ambient temperature. Readings taken from
receptacular platform centre of D-1 flowers at noon

stages of the blossom, whereas the heat is Ecological role of heat, potential rewards,
dissipated during the fully open, male period. and volatile cues
Internal air temperature at D-1 averaged 25 C
Volatilization of scent. Schneider and Bucha-
(N=7); this contrasts to the value given by
nan (1980) and Seymour and Schultze-Motel
Seymour and Schultze-Motel (1996), who
(1996) concur that the heat enhances the
reported 30–35 C from flowers in a pond in
volatilization of the odour, invoking the classic
Australia.
analogy of thermogenicity in the Araceae. As
In conclusion, the site of active heat gener-
noted above, the androecium, at least, receives
ation and the accompanying metabolic flare-up
heat by radiation and thus participates in the
is the glandular, sub-hypodermal ‘‘crust’’ of the
volatilizing effect. As an alternative or com-
receptacle and the subjacent storage tissue. In
plementary hypothesis, these authors also
the staminal appendages the epidermal epithe-
suggest that the heat provides a warm shelter
lium alone is responsible for scenting and the
to pollinators, at least in D-1 flowers and the
epithelium apparently also plays this role in the
following night(s), thus even acting as a
receptacle. Although non-thermogenous osmo-
primary attractant. For the arum lily, such
phores in which a glandular and storage mes-
an interpretation was disproved experimen-
enchyma is involved in synthesising volatiles are
tally by Knoll (1926). The hypothesis claiming
known (Vogel 1963, 1990), this pattern does not
the operation of an energetically expensive
seem to apply for Nelumbo. Accordingly,
‘‘stove’’ in a warm-temperate to tropical
Lamprecht et al. (1998) concluded that ‘‘…heat
environment appears less attractive to us than
loss is not a by-product of biosynthetic activ-
the volatilization theory. The issue also
ity’’. Nevertheless, both phenomena, heat and
revolves around the type of pollinator, on
fragrance, are functionally linked, and the
which Nelumbo depends.
occurrence of thermogenicity also strongly
Pollinators. Knuth (1904) provides the
suggests that it serves a purpose in the pollina-
only record on Old World lotus – a halictid
tion ecology of lotus.
184 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
bee – in his (otherwise partly incorrect)
description of the flower from Java, and
Snigirevskaja (1964) noted ‘‘bees and wasps’’
as visitors in the Caspian region, we failed to
locate any other relevant record on Old World
lotus. However, ample lists of flower visitors of
the American subspecies exist. The first was
presented by Robertson (1889) from Illinois,
enumerating 30 spp. of three insect orders.
From Texas, Sohmer and Sefton (1978)
recorded 70 spp., members of 53 families and
10 orders, and Schneider and Buchanan (1980)
listed 25 insects determined to species, belong-
ing to four orders.
Remarkably, the comprehensive data of
these authors reveal no coincidence at the
species level between the three lists, except for
the (non-native) honey bee. Only two corre-
spondences, the coleopterans Chauliognathus
pennsylvanicus (Cantharidae) and Diabrotica
undecimpunctata (Chrysomelidae), and at gen-
eric level, nine further coincidences appear in
the two Texan populations; one genus,
(Diabrotica), corresponds in all three lists.
Most visitors were pollen-feeding or pollen-
collecting at D-2 flowers; none fed on staminal
appendages and none was found imprisoned in
closed floral chambers in the first night. Honey
bees, halictid bees and syrphids were also seen
probing the stigmata with their mouth parts by
Schneider and Buchanan (1980). No doubt, a
number of visitors transfer pollen by crawling
around in D-2 flowers, effecting autogamy in
D-2 flowers (when receptivity and pollen issue
overlap) or xenogamy.
While Robertson (1889) regarded andrenid
bees and syrphids to be principal pollen
vectors, Sohmer and Sefton (1978) indicated,
besides halictid bees and hover flies, beetles of
the genus Diabrotica as the most important.
Schneider and Buchanan (1980) also noted
halictids, but emphasized the role of coleop-
terans (Chauliognathus) – at least in some of
the Texas stands. Note that Robertson in a
later paper (1890) described two bees, Ephyla-
eus (Halictus) nelumbonis (Halictidae) and
Hylaeus (Prosopis) nelumbonis, as oligotropic
to Nelumbo in Illinois. This appears to be the
only specific relationship. These bees are
secondary and local pollinators at best: they
have a short flight period, whereas their host
has a blooming period of up to three months.
Moreover, they restrict their activity to the
harvest of pollen, thus paying visits to D-1
flowers only casually. Their adaptation to our
plant is presumably unilateral and secondary.
In summary, we are dealing with a mixed
assemblage of insects involved; most of these
appear to behave as opportunists and none of
them reveal a particular relationship to the
elaborate floral design and course of anthesis
of this plant. Considering the empirical data, is
it, then, justified to follow Bernhardt’s (2000)
classification of the Nelumbo flower as a
generalist?
Rewards. The first step is to critically
examine the flower’s potential offerings as a
primary attractant: pollen, stigmatic sub-
stance, and staminal appendages, not counting
shelter. The significance of surplus pollen is
undisputed, though only D-2 flowers offer
pollen. Records on nectareous fluid of the
stigmata, reportedly licked by some visitors,
are rather inconsistent. Meeuse (1990), for
example, claimed the ‘‘stigmatic fluid’’, besides
pollen, as the most important floral reward of
lotus. He even termed it a kind of pollination
drop, possibly derived from those of gymno-
sperm ancestors. Sohmer and Sefton (l.c.)
mention a ‘‘sweet secretion’’ of the stigma
and Igersheim and Endress (1998) noted a
‘‘secretory surface of stigma’’. Other authors
speak of ‘‘moist appearance‘‘ (Schneider and
Buchanan, l.c.) and ‘‘mucilagineous secretion’’
(Seymour and Blaylock 2000). Gupta and
Ahluwalia (1977), in their detailed study of
the ovary, also stated that the stigma bears a
thick cover of mucilage. They regarded it to be
a secretion of the epithelium of long, unicellu-
lar hairs. This mucilage not only lines the
stigmatic plate but also continues through the
stylar canal onto the ovule and fills all these
gaps. Our own examination indicates, that the
continuous cover originates by fusion of the
swollen, gelatinized cell walls of the trichomes
rather than representing a true secretion.
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
We never encountered the stigmatic surface
being covered with a liquid, except for a few
microscopic oily droplets on the mucilage.
Previous observers were apparently deceived
by the glossy, strongly reflecting stigmatic
cover. Insects may be similarly deceived. In
reality, the mucilage actually has a dry though
very soft skin. The supposition that visitors are
rewarded with nectareous fluid is certainly
erroneous.
Pollen probably first adheres by pollenkitt
(not lacking in Nelumbo as held by Zetter and
Keri, cit. by Meeuse 1990). Pollenkitt becomes
apparent, when it forms yellow droplets in
chloral hydrate. The grains seem to locally
liquify the jelly, and drawings of Gupta and
Ahluwalia (1977) suggest that the grains sink
into the medium until they germinate.
The staminal appendages are the third
possible reward. Their organ-like appearance
and starch freight indicate some destiny
beyond only scent emission. Since the starch
supply largely persists, an auxiliary role con-
nected with anther nutrition can be excluded.
The nutritious content of the appendages
suggests that they represent food bodies. Their
pronounced exposition and accessibility in D-1
flowers strongly supports this view, already
advocated by Sohmer and Sefton (1978) and
Schneider and Buchanan (1980). The circum-
stance that both teams of observers never
found them removed or gnawed, remains a
serious obstacle to this hypothesis. An exhaus-
tive survey of undisturbed natural Old and
New World stands could yield a verification.
Sohmer and Sefton (l.c.), considering the age
of the taxon and lack of a recent partnership
revealing mutual adaptation, attribute to the
bodies tentatively the status of an ancient
residual that no longer finds its proper clients
among the existent assemblage of co-visitors.
Accordingly, the flower may, indeed, represent
a living fossil that survives with the help of a
surrogate of its original relationship.
Food bodies are frequent in cantharophil-
ous flowers. The staminal appendages of
Calycanthus, well known as food bodies, are
a nice parallel to those of Nelumbo. The notion
185
that lotus is basically adapted to pollination by
coleopterans (although today it depends on a
multitude of opportunists including beetles),
goes back to Delpino (1873/74), who classified
the flower among his ‘‘tipo magnoliaceo’’: ‘‘…
é un apparecchio senza dubbio cantarofilo’’.
Whereas Robertson (1889) contested this
typification, recent American observers, albeit
with some reservations, do not dispute Delpi-
no’s assumption, also shared by Willemstein
(1987). Bernhardt (2000), although terming the
lotus a generalist flower, classified it as a
combination of his cantharophilous subtypes
‘‘painted bowl’’ and ‘‘chamber blossom’’. In
fact, the flower has many traits in common
with those of Magnolia (Thien 1974), and
Talauma (Gibbs et al. 1977). Nelumbo com-
bines most of the characteristics associated
with beetle pollination, such as actinomorphy,
polyandry, protogyny, strong aromatic fra-
grance, thermogenicity, a temporary pollina-
tion chamber, pollen shed from anthers and
collecting at the perianth floor, and presence of
putative food bodies. An uncommon deviation
is that anthesis already starts in the morning
and continues during the night and the fol-
lowing day.
Secondary attractants
Does the nature of the colour and scent of the
lotus blossom, i.e. its secondary attractants,
help specify potential adapted pollinators? The
showy, UV-absorbing perianth (Burr and
Barthlott 1993) no doubt plays an important
role, but this feature is too general (widespread
in angiosperms). Regarding the olfactory cues,
we focus here on the two dominant classes of
volatiles emitted: 1,4-Dimethoxybenzene (1,4-
DMB) and the hydrocarbons. 1,4-DMB,
mainly produced by the osmogenous epithe-
lium of the central organs of the flower, and
thus preferentially volatilized by heat, was
found to be a floral scent component in
Syringa, Hyacinthus, Cucurbita, cherry, and
several orchidaceous perfume flowers (Omata
et al. 1991). All these plants attract bees of
various groups, and Syringa also attracted
186 S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
Lepidoptera. The attraction properties of this
aromatic compound to chrysomelid beetles,
such as Diabrotica speciosa and Cerotoma
arcuata tingomariana, based on spiced artificial
baits, was demonstrated by Ventura et al.
(2000). As mentioned above, Diabrotica was
the only genus of insect visitors that occurred in
all three American stands of lotus investigated.
Although species of Diabrotica (‘‘root worms’’)
are known pollen feeders in many different
hosts (Knuth 1905) and Chrysomelidae are not
typical guests of cantharophilous flowers, the
attractive power of 1,4-DMB for coleopterans
supports the scenario outlined above.
The long-chain saturated and unsaturated
hydrocarbons, diffusely produced by the distal
halves of the petals, are less specific and more
widely distributed in floral fragrances. C14 –
C17 alkanes and alkenes have been recorded
from the scent of cantharophilous Magnolia
and Victoria (Knudsen 1993). A combined
occurrence of pentadecane, pentadecene, hep-
tadecene and heptadecadiene is reported for
the flowers and pollen of Papaver rhoeas
(Dobson et al. 1996). Dafni et al. (1990) have
shown that poppy flowers attract Amphimallus
beetles (Scarabaeidae), which feed and copu-
late in them. Beyond being found in plants,
these particular hydrocarbons are present in
the Dufour glands of ants (e.g. Morgan et al.
2003, Gokcen et al. 2002), in the defense
Fig. 4. Beetles (Cyclocephala putrida) forcing their
way through the semi-closed perianth of a Nymphaea
amazonum flower at nightfall (Pantanal, Mato
Grosso; phot. S. V.)
secretions of bombardier beetles (Eisner et al.
2001) and in venom glands of social wasps
(Sledge et al. 1999). Thus, the probability exists
that the neural cells of certain insects including
beetles recognize these carbohydrates.
One scenario would imply that coleopterans
– (assumed to be the characteristic pollinators)
– are lured throughout D-1 and D-2 stages;
those entering on D-1 stay in the flower, feed on
the appendages, thereby depositing pollen on
the stigmata. D-1 entrants remain overnight in
the closed chamber and may eat pollen during
the next morning before they leave. Those
attracted on D-2 may eat pollen and dust the
still receptive stigmata with foreign or self
pollen, while the appendages become less
accessible to them. The circumstance that
advertisement is set forth nocturnally while
the perianth is almost closed, appears a paradox
at first glance (although a small mouth is
usually kept open on top and allows the
internally produced scent to escape). However,
irrespective of body size, beetles manage to
force their way between rather tightly overlap-
ping petals, as nitidulids do in Calycanthus
(S.V., unpubl.). In nocturnal waterlilies such as
Nymphaea amazonum (Prance 1980, Vogel,
unpubl.) the flowers were impetuously occupied
that way by Cyclocephala beetles and relatives
already at dusk before the flower opened at
nightfall (Fig. 4).
Concluding remarks and outlook (parts I–III)
The present contribution, along with the two
other parts (Vogel 2004, 2004), attempt to
improve and critically survey our knowledge
of this remarkable waterplant in three points:
the aeration system, the modified stellate
crystal druses, and the floral life including
heat production and scent emission. The
sacred lotus is one of the most ancient
angiosperm fossils dating from the mid-Cre-
taceous (Albian, 112 my ago). It encompasses
archaic features as well as surprisingly
advanced, sophisticated adaptations. Primi-
tive traits include the dichotomous submar-
ginal nervature of the leaves; an initially open
S. Vogel and F. Hadacek: Functional anatomy and biology of Nelumbo
stylar canal with a continuous papillose
conductive path leading from the ovule up
to the stigma; polymery of the floral organs
and choricarpy. On the other hand, several
traits appear as derived evolutionary achieve-
ments: A ventilation system driven by
thermo-osmosis, enabling the plant to grow
in oxygen-deficient mud; endurance and prop-
agation by rhizomatous storage tubers; a
peculiar floral receptacle achieving buoyancy
postflorally as a dispersal unit; persistence of
seed germinability over centuries (Wester
1973, Shen-Miller et al. 2002); a relatively
short, two-phase anthesis accompanied by a
scheduled sequence of movements; receptacu-
lar and androecial osmophores; production of
heat with compensative thermoregulation that
enforces scent emission; and presentation of
(potential) distinct food bodies. It was prob-
ably this combination of an ancient bauplan
with progressive properties that have allowed
Nelumbo to persist over aeons in changing
land masses, climates and biotic environ-
ments.
Among the unresolved issues deserving
future inquiry are determining the point of
reversal of the gas stream in the rhizome, the
ultrastructural aspects of crystal development
in the external receptacular idioblasts, and the
verification of the assumed cantharophily of
the flowers. Concerning the scent emissions of
the flowers, the extent of variation in the
single classes of volatiles that have been
detected in the the lotus flower scent is to
be assessed on a scale comparable to our
investigation of the Viennese colony. Con-
cerning the hydrocarbons in the scent emis-
sion of the petals, elucidating the correct
position of the double bonds in the detected
isomers of the unsaturated aliphatic hydro-
carbons and subsequently determinating their
recognition spectrum by insects via GC-EAG
studies might shed light onto a possible
function of these hydrocarbons as pollinator
attractants.
S.V. owes thanks Dr. G. Hausner (Mainz) for
providing provisional gas-chromatographical scent
analyses. A. Urban operated the freezing micro-
187
tome, S. Sontag and S. Pamperl kindly assisted in
various practical problems. Suggestions given by
Prof. Dr. A. Weber and anonymous referees, as well
as the stylistic improvement of the manuscript by
Dr. M. Stachowitsch are gratefully acknowledged.
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Addresses of the authors: Stefan Vogel (e-mail:
Stefan.Vogel@univie.ac.at), Franz Hadacek
(e-mail: Franz.hadacek@univie.ac.at) Institut für
Botanik der Universität Wien, Rennweg 14, 1030
Vienna, Austria.