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Organisms Diversity & Evolution
ISSN 1439-6092
Org Divers Evol
DOI 10.1007/s13127-013-0131-9
Nectar production in the pollen flower
of Anemone nemorosa in comparison
with other Ranunculaceae and Magnolia
(Magnoliaceae)
Claudia Erbar & Peter Leins
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ORIGINAL ARTICLE
Nectar production in the pollen flower of Anemone nemorosa
in comparison with other Ranunculaceae and Magnolia
(Magnoliaceae)
Claudia Erbar &Peter Leins
Received: 5 December 2012 /Accepted: 27 February 2013
#Gesellschaft für Biologische Systematik 2013
Abstract The observation that the flowers of Anemone
nemorosa offer nectar to pollinating bee-flies (Bombylius
major) prompted this investigation into the site of nectar
secretion and nectary tissue. To allow comparison on a
broader basis, other nectar-secreting pollen flowers of the
Ranunculaceae and Magnolia (Magnoliaceae) were includ-
ed in the analysis. The contradictory information available
on the function of the mouthparts of bee-flies during nectar
and pollen feeding motivated us to investigate the proboscis
structure in detail by SEM. Our investigations in Anemone
nemorosa proved, for the first time, nectar secretion in the
genus Anemone s.s. (i.e. other than the Pulsatilla group) and
in addition, within the family, a new type of a carpellary
nectary. The latter is an epithelial nectary involving the
whole epidermis of the ovarian part of the carpel. The
nectary of Anemone nemorosa resembles that of Magnolia
(e.g. M.stellata), which we re-investigated. In both
Anemone nemorosa and Magnolia stellata, nectar produc-
tion is limited mainly to the female phase of the
proterogynous flower. In this way, the attractiveness of the
flower is also assured in the non-pollen presenting phase.
Especially in Magnolia, with its numerous carpels arranged
on a cone-like receptacle, the economic disadvantage of a
choricarpous- compared to a coenocarpous-gynoecium is
compensated for by nectar secretion by each carpel. When
licking up the nectar droplets from the carpel surfaces,
contact of the insect's body with each stigma may be
achieved.
Keywords Nectaries .Anemone nemorosa .Clematis .
Caltha .Pulsatilla .Ranunculus .Ranunculaceae .
Magnolia stellata .Magnoliaceae .Proboscis of Bombylius
major
Introduction
In April 2010, in a beech forest near Heidelberg (Reilingen),
we observed bee-flies, Bombylius major,visitingflowersof
the wood anemone, Anemone nemorosa (Ranunculaceae).
This was not a singular event; several individuals of
Bombylius visited eagerly different flowers of the population
of Anemone nemorosa.Inbetween,Bombylius visited Vinca
minor (Apocynaceae), where the insect is rewarded by copi-
ous nectar. The long mouthparts of bee-flies reach deep into
the corolla tube when collecting nectar. But what is the reward
in the Anemone flower? We never expected a bee-fly on
Anemone,becauseAnemone is thought to be a true pollen
flower and, at first sight, the mouthparts of Bombylius do not
seem well suited to true pollen flowers. Small beetles, flies
and bees are often recorded as visitors of Anemone nemorosa
(e.g. Knuth 1898;Hegi1912; Proctor et al. 1996).
We never observed bee-flies taking the anthers of
Anemone nemorosa between their labella (the two distal
parts of the labium) or handling the anthers with their fore-
legs. Instead, the bee-flies probed at the bottom of the
flower, and this behaviour prompted our search for nectar
and the site of nectar production in Anemone nemorosa.
It is well-known that, within the Ranunculaceae, there are
flowers that offer only pollen and others that offer both pollen
and nectar as rewards to their pollinators. Nectar is either
secreted by special nectary organs (staminodes/"petals", e.g.
in Ranunculus and Aquilegia,e.g.Hiepko1965; Erbar et al.
1999), or at the base of filaments in some members ofClematis
C. Erbar (*):P. Leins
COS, Biodiversity and Plant Systematics, Universität Heidelberg,
Im Neuenheimer Feld 345,
Heidelberg, Germany
e-mail: claudia.erbar@cos.uni-heidelberg.de
Org Divers Evol
DOI 10.1007/s13127-013-0131-9
Author's personal copy
(Kratochwil 1988)andPulsatilla (Daumann and Slavikova
1968) and in one case (Caltha) by the carpel flanks (patches
of hairs on both flanks; Sprengel 1793;Petersenetal.1979).
For the purpose of comparison, we re-investigated members of
the above-mentioned ranunculaceous genera as well as a spe-
cies of Magnolia (M.stellata). To date, in some of the taxa
mentioned, although the site of nectar presentation is known,
detailed morphological-histological studies are lacking.
Materials and methods
Flowers from fresh or liquid-preserved material were stud-
ied. Vouchers of the collected material have been deposited
in the herbarium of the Botanical Garden of the University
of Heidelberg (HEID).
Flower buds were fixed in FAA (formalin, acetic acid,
alcohol). For samples studied using SEM techniques, the
buds were dehydrated in dimethoxymethane, critical-point
dried using liquid CO
2
, mounted on stubs, coated with gold
and studied in a Leitz (AMR 1200B, Leitz, Wetzlar,
Germany) scanning electron microscope (software: Digital
Image Processing System 2.6). For sections examined using
light microscopy (Zeiss, Software: AxioVision 4), the
flowers were dehydrated in an alcohol series, transferred to
infiltration medium and embedded in a methacrylate resin
(Technovit 7100, Kulzer, http://www.kulzer-technik.de).
The resin blocks were cut with a rotary microtome (using
disposable blades) at a thickness of 6 μm, and the tissue
sections were then stained with toluidine blue.
Material investigated is shown in Table 1.The
mouthparts of Bombylius major (Figs. 10–15)were
investigated from individuals sampled on 20 May 2012
at the border of a floodplain forest near Heidelberg
(Ketsch, Germany); these individuals visited Glechoma
hederacea (Lamiaceae).
Results
Bombylius major
In spring 2010 (19 April), between 12.30 noon and 2.30 pm,
in a beech forest near Heidelberg (Reilingen) we observed
the major bee-fly, Bombylius major, visiting flowers of
Anemone nemorosa (Figs. 1–4). Several individuals of
Bombylius moved rapidly from flower to flower in the
relatively dense population of Anemone nemorosa.
Bombylius usually hovers next to the plant, then approaches
the flower and rests with its legs on the flower to stabilize its
hovering when feeding (Figs. 3–4). When the insect holds
onto the flower, it buzzes more or less constantly with its
wings. Only occasionally, when flower-visits last some
time, is the buzzing briefly interrupted (Fig. 1). The insects
visited the white, proterogynous flowers in the female and
the early male as well as in the late male phase of anthesis.
In between, Bombylius visited the blue-violet flowering
Vinca minor (Apocynaceae; Fig. 6)andViola riviniana
(Violaceae). In both, the insect is rewarded by copious
nectar. The long mouthparts of the bee-flies reach deep into
the corolla tube of Vinca and the spur of Viola. During the
period of observation (between noon and early afternoon)
no liquid nectar could be observed in the Anemone flowers.
Nevertheless, occasionally glistening of nectar drops (in
Table 1 Material investigated by scanning electron microscopy (SEM)
Ranunculaceae:
Anemone nemorosa L. Erbar 2010 Reilingen (Germany = G) Figs. 1–5
Erbar 26/2010 (HEID 207056) Reilingen (G) Fig. 19
Erbar 24/2012 (HEID 206695) Heidelberg, Mühlental (G) Figs. 8–9,16–18,20–23
HEID 207057 Hengstbachtal (G)
HEID 207058 Nonnweiler (G)
Ranunculus aconitifolius L. Erbar 20/2006 (HEID 207112) near Ballon d'Alsace, (France) Figs. 30–35
Ranunculus acris L. Erbar 2009 Botanical Garden of the University
of Heidelberg = BGHD
Fig. 24
Pulsatilla turczaninovii Krylov and
Serg. [= P.grandis Wend. var.
turczaninovii Popov]
Erbar 31/2012 (HEID 207046) BGHD Figs. 25,36–37
Clematis alpina (L.) Mill. Erbar 1999 Italy, Southern Alps, beneath
Passo Croce Domini
Fig. 26
Wolf s.n., 2012 (HEID 207076) Austria, Steiermark, Aflenzer Staritzen Figs. 38–42
Caltha palustris L. Erbar 26/2010 (HEID 207069) BGHD Figs. 27–28,43–46
Magnoliaceae:
Magnolia stellata (Siebold and Zucc.)
Maxim.
Erbar 23/2012 (HEID 207042) BGHD Figs. 29,47–53
C. Erbar, P. Leins
Author's personal copy
limited quantity) was visible (Fig. 5, arrow), which may
occur when flowers have not been visited by insects for
some time.
The bee-flies visiting Anemone nemorosa at our study
site did not consume pollen out of the anthers. Instead, after
probing at the bottom of the flower they spread the labella
and, in doing so, they presumably dissolve solidified nectar
in saliva (Figs. 1–2).
The rigid, needle-like proboscis (ca. 10 mm long,
formed by labrum, labium and hypopharynx) is adapted
for sucking nectar deep in flowers. It is projected for-
wards more or less horizontally and cannot be retracted.
The paired labella, however, are flexible (Fig. 7)and
make initial contact with substances to be ingested. If
the labella are pressed tightly together, the proboscis is
used to suck up liquid nectar (Fig. 10). If the slender
Figs. 1-9 Major bee-fly Bombylius major visiting flowers of Anemone
nemorosa (Ranunculaceae). B.major is characterised by the dark
patches on the anterior half of the wings. Fig.2Enlarged section of
Fig.1showing the spreading proboscis when dissolving solidified
nectar. Fig.5Enlarged section of Fig.4,arrow points to solidified
nectar. Fig.6B.major visiting Vinca minor (Apocynaceae). Fig.7Tip
of the proboscis of B.major, spreading labella make the hypopharynx
visible. Figs.8–9Nectar in a just-opened flower of A.nemorosa.Hyp
Hypopharynx, La labellum
Nectar production in the pollen flower of Anemone nemorosa
Author's personal copy
labella are spread at right angles to the axis of the pro-
boscis as observed during the visits of Anemone flowers
(Figs. 1–2),thelabellaarealsoinvolvedinfooduptake
but, at the bottom of the Anemone flower, they come into
contact only with dried up nectar. Each labellum is tra-
versed on its inner side by three pseudotracheae, i.e.
canals that extend in the longitudinal axis of the proboscis
(Figs. 7,11–14). These tube-like structures have openings
for the discharge of saliva. The outflowing saliva can
dissolve the solidified nectar. The fluid is then conducted
along furrows that are formed by interpseudotracheal
folds (Figs. 13–15).
Figs. 10-15 Scanning electron microscopy (SEM) images of the pro-
boscis of Bombylius major.Fig.10 Both labella are tightly held
together forming a "drinking straw" when feeding on nectar in tubular
flowers, arrow points to solidified nectar (bee-fly caught shortly after
nectar feeding). Figs.11–14 Same proboscis at different views. Fig.11
Tip of a proboscis with spreading labella (one labellum torn off, break-
off point marked with asterisk.Fig.12 Detail of the distal part of a
labellum showing the three pseudotracheae at higher magnification.
Fig.13 Proximal part of one labellum. Fig.14 Detail of the proximal
part of the labellum showing the three convex pseudotracheae and the
alternating food furrows at higher magnification. Fig.15 Three
pseudotracheae bulged outwards with alternating food furrows, due
to the handling of the insect the zipper-like closure of the
pseudotracheae is partly broken up. FF Food furrow, Hyp hypophar-
ynx, La labellum, Lb labrum, Li labium, Pt pseudotrachea
C. Erbar, P. Leins
Author's personal copy
Flower of Anemone nemorosa and nectar secretion
Anemone nemorosa, the wood anemone or windflower, is a
long-lived perennial, and is a common, often dominant,
understorey spring herb of European (and also Asian) de-
ciduous woodland, and naturalised in parts of North
America. Its creeping rhizomes can make wide-spreading
carpets. The solitary flowers are 2 cm in diameter, with
mostly six (or seven) tepals with many stamens (mostly
about 45) and a choricarpous gynoecium (about 10–20
carpels). The sepals are usually white inside, sometimes
slightly tinged with pink outside. The flower is held erect
during the day, but closes and droops at night and in bad
weather.
Looking for nectar in the field may yield only limited
success. At midday or in the afternoon, one may sometimes
see some glistening of nectar at the base of the carpels
(Fig. 5, arrow). Only with a dissecting microscope could
we detect copious nectar in flowers that had just opened
(Figs. 8–9; flowers collected on 13 April 2012, early in the
morning).
The carpels are hairy all around their lower part
(Fig. 16). The hairs are unicellular with a somewhat
dilated base (Figs. 17–18). The epidermal cells between
the hairs are striking. Sometimes they form a group
around the base of the hair (Fig. 18). The epidermal
cells in the area of the hairy part differ from those of
the upper part of the carpel; they are smaller, more
papillate and stain (with toluidine blue) deeper due to
their dense protoplasm (Figs. 20–23). Nectar should be
secreted by the epidermis in the ovarian part of the
carpel; however, there is no underlying nectar tissue.
We were unable to determine whether the hairs also
secrete nectar. In any case, they hold the nectar.
Stomata could be found only in the stylar region above
the ovary (Figs. 19,22), where they may serve in gas
exchange. In some cases we found crystallized nectar in
the SEM preparations due to nectar rising above the
hairyregioninflowerswheremuchnectarwaspro-
duced (Fig. 19). We obtained identical results in flowers
of Anemone nemorosa sampled at four different sites in
Germany (see Material and methods).
Nectaries in other Ranunculaceae
Flowers of many members of the Ranunculaceae offer nectar
besides pollen. As examples, the different sites of nectar
secretion were demonstrated in selected species (Ranunculus
aconitifolius,Pulsatilla turczaninovii,Clematis alpina,
Caltha palustris) and for comparison in Magnolia stellata
(Magnoliaceae).
In many genera, nectar is secreted in special nectary
organs, which are formed between perianth and androecium.
These variously shaped nectary organs are often showy and
take over petal function, as for example in Ranunculus
(Fig. 24). In Ranunculus acris, like in most species of this
genus, a scale covers a nectar-secreting pit, at the base of
which nectar tissue can be found. In the white-flowering
Ranunculus aconitifolius the scale is not a flat structure but
tubular (Fig. 30). At the timewhen the stamen primordia have
already differentiated into anthers and short filaments, the
primordia of the nectary organs in Ranunculus aconitifolius
are small and flat. The primordia may exhibit slight depres-
sions (Figs. 31–32) due to pressure from contiguous stamens.
Differentiation of the nectary organs starts with the formation
of a horseshoe-shaped bulge at the ventral base of the young
nectary organ (Figs. 31–32). Soon afterwards the upwards
pointing ends come into contact, forming a ring-like structure
(Fig. 33). By further unequal upgrowth the bowl-shaped
structure changes into a obliquely tube-shaped scale whose
longer part may be entire or slightly two-lobed (Fig. 30). A
massive nectar-secreting tissue of small, plasma-rich cells lies
at the base of the tubular scale (Figs. 34–35). Nectary slits
could not be detected.
In the Siberian pasque flower, Pulsatilla turczaninovii,
plenty of nectar is secreted, mainly during the early female
phase of anthesis (flowers are proterogynous; Fig. 25). The
nectar is secreted by the outer short, club-shaped sterile
stamens (staminodes), either from the filament or from the
entire staminode (Figs. 36–37).
Clematis alpina has outer spatulate staminodes
(Figs. 26,38). However, it is not these staminodes,
but rather the fertile inner stamens that are the site of
the nectary—strictly speaking the ventral side of their
filaments (Fig. 39). Nectar is secreted by the epidermis
in an oval area (Figs. 40–42). In this area the epidermis
differs from that of the surrounding area: there are deep
longitudinal furrows between the secreting cells
(Fig. 41), perhaps capillary nectar holders.
Caltha palustris—the kingcup or marsh marigold
(Fig. 27)—exhibits a carpellary nectary. At anthesis, flowers
of Caltha have droplets of nectar between the carpels
(Fig. 28). On either side of each carpel, there is a basal
group of approximately 100 unicellular, clavate trichomes
that are responsible for nectar secretion (Figs. 43–46).
In Magnolia stellata, nectar secretion is found only at the
beginning of anthesis (the flowers are proterogynous;
Fig. 29). Small droplets are produced in the region of the
style and the ovary. There is no localised nectary tissue
below the epidermis (Figs. 50–51), but the epithelial nectary
covers the whole carpel from style (Fig. 52) to ovary
(Fig. 53). In objects prepared for SEM investigation, the
surfaces of the carpels are coated by a granular material,
presumably solidified nectar (Figs. 47–49). Nectar secretion
is through the epidermal cell wall. Stomata (Fig. 48) may
only serve in gas exchange.
Nectar production in the pollen flower of Anemone nemorosa
Author's personal copy
Figs. 16-23 Anemone nemorosa.Figs.16–19 SEM images. Figs.20–
23 Histological sections. Fig.16 Choricarpous gynoecium; note the
basal hairy part of the carpels. Figs.17–18 Hairs and epidermis at
higher magnification. Fig.19 Carpel surface above the basal hairy part;
note the crystallised nectar and the stomata presumably for gas ex-
change (arrows). Fig.20 Longitudinal section through a carpel; note
the different size and staining of the epidermal cells in the ovarian and
stylar area; arrow transition area of both parts. Fig.20 Transition area
at higher magnification. Fig.22 Cross section through the style; note
the large, ± unstained epidermal cells; arrow stoma. Fig.23 Cross
section through the ovary; note the cytoplasm-rich and thus more
intensively stained epidermal cells (carpellary epidermal nectary). O
Ovary, Ov ovule, Sty style
C. Erbar, P. Leins
Author's personal copy
Discussion
The mouthparts and their function in Bombylius major
Bombylius major (Bombyliidae) is found frequently in the
whole northern temperate zone, from Europe to parts of
Asia, and in North America. It is well-known as a nectar
feeder and, with its long proboscis, concealed nectar can be
easily exploited. However, it also visits flowers with more
or less freely presented nectar where a long proboscis is not
required. Knoll (1921: p.105) was the first to mention pollen
consumption by a Bombylius species (B.medius), and it has
been shown recently that females, at least, are obligate
pollen feeders since pollen is a necessary requirement for
nourishing developing eggs (Boesi et al. 2009).
The proboscis can be used to suck up liquids, either fluid
nectar or solid material like pollen grains suspended in salivary
secretions. Knoll (1921) did not describe how Bombylius man-
ages the uptake of pollen, and among zoologists there is discus-
sion whether and how Bombylius can consume pollen directly
from the anthers. On the one hand, it is described that the anthers
are taken between the labella and that pollen is scraped off by
rubbing and twisting movements (Schremmer 1961;Szucsich
and Krenn 2002). On the other hand, pollen collection could be
accomplished with the forelegs, which bear modified setae
(bristles) playing a role in pollen removal; the forelegs then
Figs. 24-29 Androecial nectaries in the Ranunculaceae. Fig.24 Ra-
nunculus acris, scales covering the nectary at the base of each petaloid
staminode. Fig.25 Pulsatilla turczaninovii, just-opening flower in the
early female phase of anthesis presenting abundant nectar at the base of
the androecium. Fig.26 Clematis alpina, one tepal dissected (scar
marked with asterisk). Figs.27–29 Carpellary nectaries. Figs.27–28
Caltha palustris, presenting a large nectar drop between the lateral
flanks of the carpels. Fig.29 Magnolia stellata, presenting small nectar
droplet at the stylar surface; note that in the early female phase of
anthesis the stigma is covered by a secretion. CCarpel, Sto staminode,
Ttepal. Arrows Nectar drops or droplets
Nectar production in the pollen flower of Anemone nemorosa
Author's personal copy
transfer the pollen to the tip of the proboscis (Neff et al. 2003;
see also Deyrup 1988, for another species of Bombyliidae).
It has been suggested that the pseudotracheal system in the
labella serves to distribute saliva onto the labellar surface when
feeding on solidified nectar (Krenn et al. 2005). Conversely,
however, food seems not to be transported by the
pseudotracheae as repeatedly suggested (e.g. Szucsich and
Krenn 2002; Krenn et al. 2005). Instead, the food (liquid and
solid) is conducted along furrows that are formed by
interpseudotracheal folds (Schuhmacher and Hoffmann 1982;
Gilbert and Jervis 1998), as shown by Dimmock in a simple
experiment as early as 1881. He found that, after feeding the
bee-fly with a mixture of sugar and gum arabic, coloured with
carmine, and then plunging it suddenly into alcohol to fix the
coloured solution in its mouthparts, the coloured solution of
gum arabic had not entered the pseudotracheae.
Folding of the labellar surface has been denied in
Bombylius major (see Szucsich and Krenn 2002; Krenn et
al. 2005). However, using SEM, we could demonstrate such
food furrows in two proboscises whose labella are spread:
food furrows can be seen at the proximal part of a labellum,
at the entrance to the epipharyngeal food canal (Figs. 13–15).
In a feeding syrphid (Eristalis), Schuhmacher and Hoffmann
(1982) demonstrated by instantaneous freezing that the
interpseudotracheal folds bulged outwards (due to
haemolymphal pressure) so that the pseudotracheae are at
the bottom of the food furrow. In our material of Bombylius
major (caught from the flower and not further treated by
chemicals) the folding is just reverse, namely the
pseudotracheae bulging outwards and presenting a zipper-
like closure at the edge (Figs. 13–15).
Bombylius species that feed mainly on nectar from deep
corollas exhibit only a few pseudotracheae (Gilbert and
Jervis 1998). Since at least the females depend on pollen,
saliva welling out of the pseudotracheae is necessary so that
pollen mixed with a fluid can be sopped up. Our observation
Figs. 30-35 Ontogeny of the nectary organ in Ranunculus
aconitifolius.Figs.30–34, SEM images. Fig.30 Tubular scale of an
adult nectary organ. Figs.31–33 Early developmental stages of the
tubular scale. Fig.34 Tubular nectary scale cut in half. Fig.35 Longi-
tudinal section through the nectary. Ne Nectary. Asterisk Correspond-
ing sites
C. Erbar, P. Leins
Author's personal copy
of spreading labella at the bottom of the flowers (Figs. 1–2)
allows the interpretation that solidified nectar is dissolved in
saliva and is sucked along the food furrows to the opening
of the epipharyngeal food canal.
Pollination of Anemone nemorosa by Bombylius major
More than 50 species of flowering plants are recorded to be
pollinated by Bombylius major (e.g. Knuth 1898;
Figs. 36-46 Androecial and carpellary nectaries in Ranunculaceae.
Figs.36–37 Pulsatilla turczaninovii.Fig.36 SEM image of part of
the androecium with the outer staminodes retarded in development.
Fig.37 Longitudinal section through a nectar-secreting staminode.
Figs.38–42 Clematis alpina.Fig.38 Outer staminode. Fig.39 Fertile
stamen with broadened filament. Fig.40 Magnification of the ventral
oval nectar-secreting area of the filament. Fig.41 Transitional zone
between the longitudinally furrowed nectar-secreting cells (left) and the
surrounding cells (right). Fig.42 Longitudinal section through the
nectar-secreting epidermis, upper end of nectar-secreting area indicated
by an arrow.Figs.43–46 Caltha palustris.Fig.43 SEM image of the
gynoecium. Fig.44 Group of nectar-secreting trichomes on either side
of the carpels. Fig.45 Unicellular, clavate hairs at higher magnifica-
tion, arrows indicate stomata for gas exchange. Fig.46 Cross section
through the carpel wall with the nectar-secreting cells (carpellary
trichome nectary). AAnther, Ccarpel, Ffilament, St stamen, Sto
staminode, Ttepal
Nectar production in the pollen flower of Anemone nemorosa
Author's personal copy
Graenicher 1910; Hegi 1912; Kugler 1970; Proctor et al.
1996; Kastinger and Weber 2001; Panov 2007), most of
them blooming in spring. The majority of flowers visited
are violet, purple, blue, but also white and yellow, covering
different blossom types (e.g. tubular, salverform, disc-
shaped). Anemone nemorosa cannot be found among the
flowers recorded for visits by Bombylius major (citations
see above). Although Kastinger and Weber (2001)cite
Fritsch (1927a,b), we could not find Bombylius major as
pollinator of Anemone nemorosa in the compilations of
Fritsch. But Bombylius venosus is mentioned as visitor of
Anemone nemorosa (and two further Anemone species) by
Barwisch (1938). Knight (1967), however, emphasises that
Anemone nemorosa, no matter how abundant, were never
seen by him to be visited by Bombylius species (study area
in the midlands near Warwick, UK).
Since Bombylius individuals fly early in the year (from
the end of March to June), they may be quite important as
pollinators of spring flowers. However, the range of plants
visited varies depending on the local flora (e.g. Grimaldi
1988; Panov 2007). Temperature and solar radiation deter-
mine the distribution and emergence of Bombylius major:It
will not fly in temperatures less than 62 °F (16.5 °C) or on
dull days (Knight 1967). In spring 2010, individuals of
Bombylius major were busy pollinators of Anemone
nemorosa at our observation site, visiting flowers at differ-
ent phases of anthesis. Due to the need to dissolve the dried
up nectar, they spend some time on the flowers (buzzing is
briefly interrupted: Fig. 1). When spreading the labella, they
move further down and thus come into contact with stigmas
or anthers of the slightly proterogynous flowers (Figs. 1–4).
Since, in addition to proterogyny, Anemone nemorosa is
Figs. 47-53 Magnolia stellata.Figs.47–49 SEM images. Figs.50–53
Histological sections. Figs.47–49 Surface of the carpels is covered
with solidified nectar. Figs.48–49 Stylar surface at higher magnifica-
tion; arrow stoma for gas exchange. Fig.50 Longitudinal section
through a carpel. Fig.51 Stylar region with the epithelial nectary
(right). Fig.52 Cross section through the style. Fig.53 Cross section
through the ovary. OOvary, Ov ovule, Sti stigma, Sty style
C. Erbar, P. Leins
Author's personal copy
self-incompatible (Shirreffs 1985; Müller et al. 2000), self-
fertilisation is avoided.
Most authors emphasise the importance of vegetative
reproduction by rhizome branching and fragmentation com-
paredwithsexualreproductionbyseedsinAnemone
nemorosa (e.g. Shirreffs 1985), and sexual reproduction
hasbeenconsideredtoplayonlyamarginalrolein
established populations (Tumidajowicz 1975). However,
recent studies show that the amount of reproduction by
seeds is higher than previously thought (Holderegger et al.
1998; Müller et al. 2000), and that there is high genetic
variation in the population structure, confirming the impor-
tance of sexual reproduction (Stehlik and Holderegger
2000).
Nectar in other multistaminate Ranunculaceae
and in Magnolia
Outside the Magnoliaceae and Ranunculaceae, nectar-
secreting tissue is recorded in a number of multistaminate
members of the lower organisational level of the angio-
sperms, e.g. Nymphaeaceae (Schneider et al. 2003),
Illiciaceae (Thien et al. 1983), Calycanthaceae, Lauraceae,
Monimiaceae (Endress 1992,2010), and Annonaceae
(Silberbauer-Gottsberger et al. 2003).
Ranunculaceae belong to the early-diverging eudicots and
are a "transitional" group between basal angiosperms and core
eudicots. This in-between group is diverse in its floral charac-
ters, including the site of nectar secretion. Most
ranunculaceous flowers offer nectar in addition to pollen.
The special nectary organs between perianth and androecium,
which serve in nectar production and nectar presentation, are
known widely and have been investigated developmentally in
a number of studies (e.g. Hiepko 1965;KosugeandTamura
1989; Erbar et al. 1999; Ren et al. 2009,2011;Zhaoetal.
2012a). Their shapes can vary considerably: tubular, linear-
oblong, flat(petaloid with the basal nectary pit mostly covered
with a scale) or spurred. The nectariferous tissue is always
mesophyllary. With Ranunculus aconitifolius we investigated
for the first time the ontogeny of a tubular instead of a flat
scale in a species outside the batrachian group. The results do
not contradict the ontogenetic or structural resemblance to
stamens (see Erbar et al. 1999;LeinsandErbar2010). In
Ranunculus (= Batrachium)bungei the ontogeny of the tubu-
lar scale is similar to Ranunculus aconitifolius: A horseshoe-
shaped ridge develops into a circular rim and finally into an
oblique cup-shaped scale (Zhao et al. 2012b; see also adult
scale shapes in Ranunculus subgen. Batrachium in Dahlgren
1992). In Ranunculus sceleratus (Zhao et al. 2012b), early
stages resemble those of Ranunculus aconitifolius and
Ranunculus bungei, but formation of a circular rim does not
occur, so that in adult stages the pocket-like nectary has a
horseshoe-shaped rim.
It is well-known that some members of Clematis
(Kratochwil 1988) and Pulsatilla (Daumann and Slavikova
1968) secrete nectar at the filaments; however, detailed
studies are lacking. We could confirm that in Pulsatilla (P.
turczaninovii) nectar is secreted by the outer short, club-
shaped sterile stamens (staminodes), either from the fila-
ment or from the entire staminode (mesophyllary nectary).
Within the genus Clematis, there are both pure pollen
flowers such as Clematis vitalba, in which all stamens are
fertile, and pollen-nectar flowers such as Clematis alpina
with outer spatulate staminodes. However, it is not the
staminodes, but the inner side of the fertile stamens that
are the sites of epithelial nectary.
In addition to these cases of androecial nectaries (situated
at staminodes or stamens), there is a well-known carpellary
nectary, namely in the flower of Caltha palustris, which has
been studied intensively by Petersen et al. (1979). Until this
study, Caltha was the only case in Ranunculaceae in which
a carpellary nectary was known. Its patches of hairs on both
carpel flanks differ from the nectary of Anemone nemorosa
presented here. There was a lost hint in an old textbook by
Fritch and Salisbury from 1920 that, in Anemone nemorosa,
nectar is secreted by papilla-like epidermal cells.
Nevertheless, our investigations confirm this early observa-
tion, and add a new type of a carpellary nectary within the
family Ranunculaceae. Fritch and Salisbury (1920, p. 148),
however, do not specify which floral organ secretes nectar in
Anemone nemorosa. Van Tieghem (1892, p. 432) assumed
"un nectaire diffus" in the receptacle (in both Anemone
nemorosa and Caltha palustris).
It is of interest that the site of nectar secretion differs in
Anemone nemorosa and Pulsatilla. Recent molecular data
suggest the inclusion of Pulsatilla (and further small genera)
in a large genus Anemone.Pulsatilla (Anemone section
Pulsatilla) appears to be sister to Anemone s.s. (Anemone
section Anemone; Hoot et al. 2012).
Since an epithelial ovarian nectary, which we report for
the first time in Ranunculaceae, was described by Daumann
(1930)inMagnolia species, though without satisfactory
evidence, we investigated Magnolia stellata in detail.
Nectar secretion takes place only at the beginning of anthe-
sis. As in Anemone nemorosa, there is no nectariferous
tissue below the epidermis. We assume that nectar is secret-
ed through the epidermal cell wall; we never found any
nectary slits. There may be channels in the cuticula through
which the nectar exits (as reported for the nectary of Vicia by
Gunning and Steer 1975). In contrast to Anemone nemorosa,
in Magnolia there is an epithelial nectary involving the
epidermis of the entire carpel (and not only the epidermis
of the ovary).
After preparation for SEM, the carpel surfaces of
Magnolia stellata are evenly coated with a granular sub-
stance, presumably crystallized nectar (Figs. 47–49),
Nectar production in the pollen flower of Anemone nemorosa
Author's personal copy
although distinct small nectar droplets are visible in the
flowers (Fig. 29). After fixation, we consistently observed
solidification or flocculation of substances in areas covered
by nectar in the flowers in different groups of flowering
plants. It is not known whether the granular material con-
sists of sugar or more complex chemicals. Although the
main ingredients of nectar are the three sugars sucrose,
glucose, and fructose (and other carbohydrates like maltose
and raffinose in small amounts), followed by amino acids
and proteins (e.g. enzymes like invertase), nectar contains
many other compounds, such as inorganic ions, organic
acids, vitamins, antioxidants, phenolics, alkaloids, lipids,
and terpenoids in minor concentrations (Lüttge 1961,
1962; Baker and Baker 1983; Nicolson and Thornburg
2007). Nectar chemistry may be altered by fixation with
FAA (formalin, acetic acid, alcohol, water) as used in our
preparation process. Pacini et al. (2003) emphasise that
nectar is not removed during fixation, dehydration and
staining with PAS (periodic acid Schiff).
The diverse structures, the rare occurrence, and scattered
distribution of nectaries in the basal groups indicate conver-
gent evolution. Disc nectaries, however, seem to occur only
outside the ANITA (or ANA) grade, magnoliids, monocots,
and also outside Ranunculales among basal eudicots
(Endress 2008,2010,2011). Disc nectaries, or better
receptacular disc nectaries, with the typical nectary slits
are characteristic of many eudicots. It is worth mentioning
that, in core eudicots, a transcription factor encoded by the
CRABS CLAW gene is required for nectaries that may occur
anywhere in the flower. First results in a limited number of
taxa examined (e.g. Arabidopsis,Cleome,Nicotiana,
Petunia) indicate that, irrespective of the position within
the flower, the CRABS CLAW gene is essential for nectary
development (Bowman and Smyth 1999; Lee et al. 2005a,
b). Its expression is limited mostly to carpels (in that its
ancestral function is involved in suppressing early radial
growth of the gynoecium and in promoting its later elonga-
tion) and nectaries (Bowman and Smyth 1999; Fourquin et
al. 2005). However, in basal eudicots, no evidence for
expression of this gene in nectaries could be found (nectary
spur of Aquilegia investigated; Lee et al. 2005a,b).
The evidence of nectar in Anemone nemorosa (fluid or
solidified) may also resolve the puzzle that there are reports
on insects tapping juicy tissues with the proboscis in what
were thought to be nectar-less flowers: Müller (1873)
reported a "piercing" bee-fly (Bombylius canescens) work-
ing on Hypericum perforatum and a "piercing" honey bee
on Anemone nemorosa. It was assumed (see also Bonnier
1879 and Knuth 1898) that the insect bores with its probos-
cis into the base of the flower to obtain sap from floral
tissue. However, Schremmer (1961) already pointed out that
Bombylius is not able to penetrate floral tissue with its
proboscis.
Why nectar secretion in pollen flowers?
In magnoliids, floral rewards are pollen, nectar, and food
bodies (e.g. Calycanthus, Calycanthaceae) and, in some
cases, pollination chambers (members of Annonaceae). If
nectar, mostly in small amounts, is secreted, the nectaries
are located on tepals, staminodes, stamens, or carpels (C.E.,
manuscript in preparation).
In Anemone nemorosa,Caltha palustris, and Pulsatilla
turczaninovii as well as in Magnolia stellata, nectar produc-
tion is limited mainly to the female phase of the (at least
slightly) proterogynous flower. By this, the attractiveness of
the flower is also assured in the non-pollen presenting phase
of anthesis (or early male phase with only little pollen
offered). Especially in Magnolia, with its numerous carpels
(about 40 in Magnolia stellata) on the cone-like receptacle,
the economic disadvantage of a choricarpous gynoecium
compared to a coenocarpous one is compensated by nectar
secretion of every carpel. In a coenocarpous gynoecium an
uneven pollen deposition onto the stigmata can nevertheless
result in the fertilization of all ovules due to the common
inner gynoecial space, the so-called compitum (Endress
1982; Leins and Erbar 2010). However, when licking up
the nectar droplets from all carpels in the choricarpous
Magnolia flowers, contact of the insect's body with all
stigmas might be achieved and thus, at best, fertilization of
the ovules in all carpels.
Acknowledgements We thank Peter Endress and an anonymous
reviewer for useful suggestions. We are grateful to Peter Endress and
Graham Muir for improving our language. We thank Anton Weber and
Susanne Kastinger for the reference of Barwirsch (1938).
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