560
Original Paper
Nectary Structure, Nectar Secretion Patterns and Nectar Composition in Two Helleborus Species J. L. Vesprini1'2, M. Nepi2, and E. Pacini2 Facultad de Ciencias Agrarias, UNR CC14 2123 Zvalla, Argentina 2 Dipdrtimento di Biologia Ambientale, Università di Siena, Italy Received: April 9, 1999; Accepted: July 10, 1999
Abstract: The morphological and cytological characteristics of nectaries of Helleborus foetidus and H. bocconei during the secre-
tory period are reported. The nectaries are derived from modified petals and secrete nectar continuously for about 20 days; they consist of a single layered epidermis, nectar-producing parenchyma and photosynthesizing parenchyma. Nectar secretion is holocrine and the nectar is released by rupture of the wall and cuticle of each epidermal cell. The nectaries of the two species differ in number and external morphology. In H. foetidus, secretion begins before anthesis and secretion rate decreases with nectary age. In H. bocconei it begins on the day of anthesis and proceeds at a constant rate. The nectar has a high sugar content, mainly sucrose, and also contains lipids and proteins.
thin permeable cuticle; 3) through pores in the cuticle; 4) after rupture of the cuticle under nectar pressure. Secretion is generally eccrine or granulocrine (Fahn, 19881181).
Nectar may be regarded as an aqueous solution of sugars, mainly sucrose, glucose and fructose. Amino acids and proteins (mostly enzymes) are other common constituents. Various substances that vary from species to species may be present in traces (Baker and Baker, 1983 a]1]).
Nectar composition and its secretory pattern vary between species and within the same species in relation to environmental parameters (Fahn, 19791161; Cruden et al., 198316]: Mar-
den, 19841301; Freeman and Head, 19901191; Wyatt et al., Key words: Nectary structure, secretion mechanisms, nectar secretion patterns, nectar sugar composition, Helleborus.
19921431) and physiological factors (Gottsberger et al., 1990123]; Petanidou et al., 1996136]). In species with hermaphrodite flow-
ers, variations in nectar composition may occur in relation to the sexual phases (Gillepsie and Henwood, 19941221). It was re-
Introduction
cently shown that the flower itself can modify nectar composition, even after its secretion, by reabsorption of sugars (Cruden et al., 198316]; Burquez and Corbet, 199115]; Nicolson, 1995132];
The nectary is a common secretory tissue in angiosperms; it produces nectar, a sugar solution which is regarded as one of
Nepi et al., 19961311: Davis, 1997; Koopowitz and Marchant,
1979116]). Nectaries may have different morphologies, may be situated in vegetative (extrafloral nectaries) or reproductive parts of plants (floral nectaries) and may have different anatomical origins (Fahn, 1979[6]). This is why the term nectary does not refer to a single, well defined anatomical structure. Nectaries usually consist of an epidermis and a parenchyma specialized for nectar production (Fahn, j979116]; Durkee,
The present study is concerned with nectary structure and nectar secretion and composition in Helleborus foetidus and Hellebonis bocconei, two species which flower synchronously
the main rewards for pollinators in many species (Fahn,
19821101). The cells of this tissue are generally small, with dense cytoplasm and a relatively large nucleus (Durkee, 1982110]). The
parenchyma may perform photosynthesis, providing materials for nectar production, or it may store starch from other photosynthesizing parts of the plant (Nepi et al., 19961311 and references therein).
1998129]).
in winter and are pollinated by bumble-bees (personal observations). Since these plants are not apomictic and automatic pollination does not occur, they depend on pollinators for seed production. Herrera and Soriguer (1983126]) studied inter- and intrafloral
variation in nectar production in populations of H. foetidus growing in southern Spain and found wide variations in the quantity of nectar in flowers at the same stage of anthesis. Eymé (1966113]) and Eymé and Le Blanc (19631141) studied the
ultrastructure of the nectary of H. foetidus. Other authors have Nectar may emerge from the nectary in different ways, some of
which are related to nectary anatomy. The most common of these are: 1) through stomata in the epidermis: 2) through a
Plant biol. 1(1999) 560-568 © Georg Thieme Verlag Stuttgart. New York ISSN 1435-8603
reported the type and form of these nectaries (Percival, 1965; Proctor and Yeo, 1973138]). Corbet et al. (197917]) inves-
tigated the regulation of nectar sugar concentration in several plants, including two Hellebonis species.
AU
Plant biol. 1 (1999) 561
Nectar and Nectaries in the Genus Helleborus
Materials and Methods Study area
Populations of H. bocconei and H. foetidus growing in a deciduous submediterranean Quercus cerris forest near Monteriggioni (Central Italy, Province of Siena, 43°23'38"N, 11 °40'18"E, elevation 213 m) were studied in the field and sampled during January, February and March of 1997 and 1998. Nectary structure
were obtained from flowers of both species in four stages: I) before opening of the flower; 2) on the first day of anthesis; 3)10 days after the beginning of anthesis; 4) before the nectaries fell, i.e., 20 days after flower opening. Flowers of both species are protogynous. Since the phases of anthesis varied considerably in length, flowers in stages 3 and 4 belonged to different phases: hermaphrodite, male and beyond the peri-
C
Samples
25
od of anthesis.
Samples were fixed in 5% glutaraldehyde in phosphate buffer
(pH 6.9), dehydrated in an ethanol series and embedded in Technovit 7100 (Heraeus Kulzer GmbH).
0
Fig.1 Flower and nectary structure in both species. (A) Section of a flower of H. foetidus x 1 (nectaries arrowed). (B) Front and side views of a nectary of H. foetidus x 3. (C) Section of a flower of H. bocconei x 0.8 showing nectaries (arrows). (D) Front and side views of a nectary of H. bocconei x 3.
Semithin sections were stained with: a) toluidine blue (TBO) (O'Brien and McCully, 19811331) as general stain; b)PAS for total
insoluble polysaccharides (O'Brien and McCully, 1981133]); c) auramine 0 for cuticle (Heslop-Harrison, 1977127]); d) calcofluor for cellulose (Hughes and McCully, 1975128]); e) FITC for proteins (Pearse, 1968]]).
In H. bocconei the nectar was withdrawn: one day before opening of the flower, the day of opening, and 5,8,11,14,17 and 20 days after opening. Five flowers were sampled at each time.
Statistical analysis
To observe photosynthesis by nectaries, thin hand-cut sections (20—50 m thick) were examined by fluorescence microscopy for autofluorescence of chioroplasts.
The variables volume, concentration and total sugars had a
Pattern of nectar secretion
(Statsoft, 1993141]).
Before anthesis commenced, 40 flowers of H. bocconei and 36 of H. foetidus were bagged with voile bags to exclude pollina-
Nectar composition
tors. Different treatments were randomly assigned to each
In both species, sampling for the determination of sugars was carried out during the following floral phases: female phase, hermaphrodite phase, male phase. Nectar samples were stored
flower, the treatment consisted of the removal of nectar at different times after flower opening. Each flower was sampled only once at a fixed age by removal of nectar. Where young flowers were sampled (up to 15 days old), these flowers were resampled 10 days later.
The nectar was collected with graduated micropipettes from all nectaries of each flower. The volume of nectar from each flower was recorded and the sugar concentrations measured by means of a refractometer (0—90 Brix). These two parameters enabled us to calculate sugar production, as described
normal distribution and were therefore processed by regression analysis using the programme Statistica for Windows
at — 80°C
until analysis. Sucrose, D-glucose and D-fructose concentrations were determined with Boehringer Mannheim kit no. 716260 based on spectrophotometric determination of enzyme reduction of NADP at 340 nm. Before measurement, the samples were diluted 1: 300 with distilled water. Standard solutions of carbohydrates were measured as controls. The results were expressed in mg/ml of nectar. Lipids and proteins were identified by spot staining methods
by Bolten et al. (1979]]).
as described in Dafni (199218]).
In H. foetidus, the nectar was withdrawn at the following times: one day before opening of the flower, the day after
Results
opening, and 5, 10, 15 and 20 days after opening. Six flowers were sampled at each time. On day 20, part of the nectar was found on the tepals, and since it could not be collected, the
data for this time point were excluded from the statistical analysis for this species.
Nectary morphology
The nectaries are inside a corona of five green tepals. In H.foetidus, the flower is bowl-shaped and the nectaries are not di-
rectly exposed (Fig. 1 A). In H. bocconei, the flower is cupshaped and the nectaries are more exposed (Fig. I C). In both species, the nectaries are green and show the typical red fluo-
562 Plant biol. 1 (1999)
Fig.2
J. L. Vesprini, M. Nepi, and E. Pacini
Plant biol. 1 (1999) 563
Nectar and Nectaries in the Genus He!Ieborus
rescence of chlorophyll when observed by microscope under UV light. They are attached to the receptacle by a stalk which
is more evident in H. bocconei and shorter in H. foetidus (Fig.1). In the latter, the nectaries are tubular (5 x 1.5 mm) and the distal part has a toothed border and is open (Fig. 1 B). In H. bocconei the nectaries are more or less conical with an expanded distal part (3 mm) having a lip closing the nectary orifice (Fig. I D). In both species, the nectar-producing parenchyma is in the proximal area. The mean number of nectaries was 10.45 per flower (n=35; SD=3.31) in H. bocconei and 4 per flower
(n=35; SD=0.89)inH.foetidus. Cytology of H. foetidus nectaries
nectary structure is characterized by an external photosynthesizing parenchyma, a nectar-producing parenchyma The
two layers and the cells may be isodiametric, cylindrical or fusiform (Fig. 2D). In stage 3, a few empty cells shaped like hourglasses are observed in the epidermis, next to cells with convex walls (Fig. 2E). At this stage, the regular organization of the groups of four cells is lost in the parenchyma underlying the epidermis. In stage 2, 3 and 4, the wall and overlying cuticle of the secretory cells contain breaks (Fig.2H).
Nectar secretion in H. foetidus Nectar secretion occurs via the inner epidermis and secretory
activity is not simultaneous in all epidermal cells, but occurs in single cells in an apparently random fashion. In stage 1, secretory cells are present in the proximal part, but as secretory activity proceeds (stages 3 and 4), secreting cells appear in more distal positions.
arid an internal epidermis (Fig. 2A). The nectar-producing par-
enchyma consists of small, more or less isodiametric, polyhedral cells without intercellular spaces (Fig. 2). The cell walls contain small amounts of cellulose and are situated in the proximal part of the tubular nectary between the vascular bundles
from the stalk (Fig. 2A). The parenchyma tapers distally, so that it is V-shaped. The red autofluorescence of chlorophyll is only observed in the outer cell layers of the parenchyma. The internal epidermis covering the nectar producing parenchyma consists of a single layer and is without stomata. In stage lit consists of uniform cylindrical cells, the longitudinal axes of which are disposed perpendicular to the nectary surface in the proximal part, and parallel to it in the distal parts. In the epidermal layer, the occasional cell overlaps with others (Fig. 2B). The cuticle covering the epidermis is uninterrupted (Fig. 2C). In the parenchyma, especially in the more superficial layers, cells are arranged in a regular pattern of groups of four. This pattern is lost in the deeper parenchyma (Fig. 2B).
In stages 2 and 3, the frequency of overlapping epidermal cells
increases and their morphology is more heterogeneous until stage 4, when the epidermis of the proximal part consists of
The walls of secretory cells bulge slightly into the nectary cavity just before secretion begins (Fig. 2B). The secretion emer-
ges from the apex of the bulge due to rupture of the wall (Fig. 2F) and accumulates under the cuticle where it forms a protrusion (Fig. 2G) which grows until it bursts (Fig. 2H). The
wall and the cuticle then collapse, covering the space previously occupied by the secreting cell (Fig. 2E). The secretory material is weakly positive toTBO, FITC and PAS(Fig.2H). Cytology of H. bocconei nectaries Parenchyma structure is similar to that of H.foetidus (Fig.3A).
In the superficial layers, autofluorescence of chlorophyll is much stronger than in H.foetidus. The epidermis covering the parenchyma in stage 1 consists of a single layer and is more disorganized in the proximal part in later stages (Fig. 3B).The cuticle overlying the epidermis is uninterrupted in stage I and develops breaks in stages 2 (Fig.3C), 3 and 4. The secretory cells become club-shaped and protrude into the nectary cavity (Fig. 3D); their walls appear stretched and lacerated in PAS and TBO stained sections (Fig. 3D). Except in the layers close to the internal epidermis, the nectar-
Fig. 2 Nectary of H. foetidus. (A) Stage 2, magnification x 60, PAS. The inner epidermis (ie) surrounding the nectar cavity (nc) consists of a single layer and the cells have their major axis perpendicular to the nectary surface in the basal part. The nectar-producing parenchyma (np) is situated between the vascular bundles (vb) and the in-
ternal epidermis (ie), and consists of small cells. Photosynthesizing parenchyma (pp) is located between external epidermis (ee) and vascular bundles (vb). (B) Stage 2. magnification x 150, PAS. As secretion proceeds, overlapping cells appear in the epidermis (arrow). In the parenchyma, especially in the more superficial layers, cells are arranged in a regular pattern of groups of four. This organization is lost deeper in the tissue. The wall of the secreting cell bulges into the nectary cavity (arrowhead). (C) Stage 2, magnification x 65, auramine 0. The cuticle covering the epidermis is continuous. (0) Stage 4. magnification x 175, TBO. The internal epidermis (ie) in the basal area has lost its single-layered structure and the cells have heterogeneous morphology. (E) Stage 3, magnification x 230, TBO. The occasional empty cell which has already secreted nectar is visible in the epidermis. The space it leaves has the form of an hour-glass due to encroachment by adjacent cells. (F) Stage 2, magnification x 300, PAS. Secretory activity begins with bulging of the wall protruding into the nectary cavity. (G) Stage 2, magnification x 300, PAS. The wall then ruptures and the secretion builds up under the cuticle. (H) Stage 2, magnification x 300. PAS. The cuticle breaks and the secretory material is released.
producing parenchyma and the photosynthesizing parenchyma contain amyloplasts which are most abundant in stage 2 and rare in stages I and 4 (Fig. 3A). Secretion in H. bocconei
The secretory material emerges due to rupture of the individual cell walls (Fig. 3E) and then the cuticle (Fig. 3C), which has interruptions where secretion has occurred. The secretory material is weakly positive to TBO, FITC and PAS. The spaces re-
maining between epidermal cells after secretory activity has occurred are smaller than in H. foetidus because the secretory cells protrude into the nectary cavity (Fig.3B). The temporal sequence of activity of the secretory cells is similar to that observed in H.foetidus. Pattern of nectar secretion In H. foetidus, nectar secretion begins before the flower opens (Fig.4) and continues for about 20 days, after which the nectaries fall. The longer the flowers were left before the first collection of nectar, the greater were the volume and concentration, and hence sugar content (Fig. 4). The total volume of nec-
J. L. Vesprini, M. Nepi, and E. Pacini
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564 Plant biol. 1 (1999)
Fig. 3 The nectary of H. bocconei. (A) Stage 2, magnification x 40, PAS. The nectar-producing parenchyma (np) is visible under the epidermis (ie) between vascular bundles (vb). Amyloplasts are visible deep in the parenchyma and in the outermost cells at the base of the nectary. (B) Stage 3, magnification x 130, TBO. The epidermis in the basal area is no longer single layered, the cells are irregular and there are empty intercellular spaces (asterisk). (C) Stage 2, magnifi-
cation x 75, auramine 0. The cuticle of bulging cells is thinner (arrow) and is broken where secretion has occurred (arrowhead). (D) Stage 2, magnification x 200, PAS. The secreting cells are clubshaped and protrude into the nectary cavity (nc); the walls are lacerated. (E) Stage 4, magnification x 200, TB0. Secretion occurs by rupture of cell wall and cuticle.
tar, sugar concentration and rate of sugar production showed a
In H. bocconei, nectar secretion begins when the flower opens (Fig. 6) and lasts about 20 days. Again, the longer the flowers were left before nectar collection, the greater were the volume and concentration, and hence sugar content (Fig. 6). Volume
negative quadratic increase with time (Fig. 4).
After 10 days, the volume decreased significantly (Fig.5A), whereas concentration did not show any significant regression (Fig. 5B). The rate of sugar production therefore decreased with increasing nectary age (Fig. 5C).
and concentration showed a quadratic pattern in relation to time, negative in the case of volume and positive for concentration (Figs. 6A,B). Hence, the rate of sugar production had a
second order regression with a linear principal component (Fig. 6C). This means that in nectaries of different ages that
Plant biol. 1 (1999) 565
Nectar and Nectaries in the Genus Helleborus A
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Fig. 4 Nectar secretion by H. foetidus during the life of the nectary. Secretion begins before anthesis and ends about 20 days later. Since part of the nectar was found on the tepals on day 20, the data at this time point were exduded from the analysis. (A) The volume of nectar secreted increased with the age of the nectary. However the increase had a negative quadratic component. The rate of nectar secretion, therefore, decreased with the age of the nectary. (B) Nectar concentration showed the same pattern as volume. The increase may be due to water loss. (C) Sugar production, therefore, had the same pattern as concentration and volume. The quantity of sugars produced increased with age of the nectary, but rate of secretion was inversely related to age of nectary.
10
15
20
Age of the nectaries
16
Fig. 5 Nectar secretion over a 10-day period in H. foetidus nectaries
of different age. (A) Volume decreased with time; the rate of decrease was positive quadratic. Older nectaries secreted less nectar than young ones in the same interval of time. (B) Concentration did not vary with age of nectary, indicating that water loss is proportional to exposure time. (C) Sugar production, therefore, decreased with age of the nectary.
Nectar composition Sucrose was the main nectar sugar in these species and nectar
composition did not change in the different phases of the life of the flower. Fructose and glucose constituted less than 5% of the sugar, and were completely absent in some samples. Mean
secreted nectar for the same period of time (10 days), sugar content is almost the same (Fig. 7C). Similarly, concentration and volume did not show any significant regression
sucrose concentrations were high in both species, being
(Figs. 7A,R).
The nectar of both species stained positively for proteins and lipids.
have
607.81 mg/mI in H.foetidus and 275.18 mg/mI in H. bocconei.
566 Plant biol. 1 (1999)
j. L. Vesprini, M. Nepi, and E. Pacini
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Fig. 6 Nectar secretion by H. bocconei during the life of the nectary. Secretion began at anthesis and ended about 20 days later. (A) The volume of nectar found increased with the age of the nectaries. The increase had a negative quadratic component, presumably due to water loss by evaporation. (B) Concentration increased positively and in an opposite manner with respect to volume. (C) The pattern of sugar production, therefore, had a dominant linear component. The rate of secretion did not change throughout the life of the nectary, indicating constant sugar production.
.
20 Age of the nectaries
Fig. 7 Nectar secretion over a 10-day period in H. bocconei nectaries
of different ages. (A) Volume decreased slightly in older nectaries (not statistically significant). (B) Concentration did not vary with age of nectary, indicating that water loss was proportional to exposure time. (C) Sugar production, therefore, did not decrease with age of nectary, confirming that production is constant in time.
taries ofSambucus nigra, secretion is not holocrine but follows the merocrine model, with the cells dying at the end of the
secretory process. Discussion Nectar secretion
manner of secretion of the floral nectaries of the two species of Helleborus is unusual. Nectar secreting cells normally remain integral throughout the period of secretion and secretion is merocrine (Fahn, 1988118]). Only in rare cases, all of which have been in extrafloral nectaries, has lysogenic secretion been reported (Fahn, 1987117] and references therein). However, Fahn (19871171) underlined that in the extrafloral necThe
If secretion involving emptying of epidermal cells is postulated, one would expect to find an epidermis with many intercellular spaces and a cuticle with many breaks in stages 3 and 4. However, few intercellular spaces were observed and the cuticle was more or less intact. It therefore seems likely that the loss of cells derived from this mode of secretion is compensated by reorganization of the cells of the nectar-producing tissue, facilitated by the relative lack of cellulose in the walls, and by repair of the cuticle. Self-repair of this type is necessary in view of the long period of activity of the nectary.
Nectar and Nectaries in
the Genus Helieborus
It is not clear how the empty spaces are filled. Two hypotheses
are: i. that adjacent cells gain space by occupying the empty spaces (Fig. 3D); ii. that the inferior cell is pushed by underlying ones into the space left by the empty cell (Fig. 2 E). The second, which seems less likely, would explain the loss of organi-
zation in the deeper tissues in advanced phases. Nectar production Materials for nectar production may be derived from substan-
ces stored during the presecretory phases, or from substances photosynthesized by the nectary itself or by other parts of the flower or plant (Fahn, 19791161). An abundance of reserve substances is generally stored when a large amount of nectar is produced in a short time (Nepi et al., 1996131]). In extrafloral nectaries, which generally produce nectar for longer periods than floral ones, starch is not normally stored and the nectaries often perform photosynthesis (Baker et al., 197813]; Durkee, 1982110]; Eleftheriou and Hall, 19831111; Fahn, 19871171; Grout and Williams, 19801241; Galetto and Bernardello, 1992 a1201; Vinoth and Yash, 19921421). The floral nectaries of the two Helle-
borus species can be regarded as "slow producers" according to the classes of Cruden et al. (198316]). They perform photosynthesis, which suggests that at least part of the nectar is produced "in situ". Our data does not allow us to estimate how
much nectar is produced by the nectary and how much by photosyrithesizing parts. However it seems likely that the nectary contribution would be the larger in H. bocconei because: 1) the flower is more open and the nectaries more exposed to light; 2) nectar is offered as soon as the flower opens, that is, when the nectaries are exposed; 3) autofluorescence of chlorophyllis more intense than in H. foetidus; 4) sugar production
is constant over time; 5) small quantities of starch were observed in the cells of more superficial parts of the nectary, closer to vascular bundles.
Plant biol. 1 (1999) 567
Nectar losses on day 20 were due to the position of the flower, which faces the ground, and to nectary structure. In this species, the nectary is open, so that when nectar volume is considerable, it may drip out of the nectary. In H. bocconei, the concentration and volume of nectar secreted followed the opposite pattern. Again, evaporation caused a positive increase in concentration and determines a negative increase in the volume over time. The increase in concentration was different from that in H. foetidus. The reasons may be that the nectar is more dilute than that of H.foetidus, especially during the first days of secretion, and it therefore loses water more easily. The flower of this species is more open and hence the nectar is more subject to the effects of the environment, despite the lip protecting the nectary opening. The effects of flower morphology in determining microclimatic protection of the nectar is demonstrated by Corbet et al. (197917]) in H.foetidus and H. lividus Aiton ssp. corsicus (Willd.) Tutin and other
plant species. Herrera and Soriguer (1983126]) report that, in populations of H. foetidus growing in southern Spain, the nectaries secreted nectar from the beginning of the female phase until half way through the male phase, when they fell. Since the duration of anthesis is variable (10—26 days) and temperature-dependent, nectar secretion may cease before anthesis ends or may continue beyond the period of pollen exposure. In fact, we sometimes observed flowers which had lost their anthers and were
forming fruit, but which had active nectaries still visited by pollinators. Pollinators and nectar composition Sugar composition, nectar volume (Baker and Baker, 1983 b121)
and flower structure (i.e., Faegri and van der Pijl, 1979115]) may
be related to the type of pollinator that visits the flower. The Pattern of nectar secretion
nectar of H.foetidus and H. bocconei consists largely of sucrose,
The duration of floral anthesis varies according to species (Pri-
our data are in accordance with the finding of Corbet et al.
mack, 198511). In the case of long-lived flowers, the nectaries may produce nectar over a limited period, the nectar remains in the flower until it is taken by pollinators; but may wait for a
(1979171). The flowers are visited predominantly by Bombus terrestris and other species of bumble-bees. Baker and Baker (1983b12]) report that long-tongued insects such as bumble-
long time, as in certain orchids (Endress, 19941121). The nectaries of our Helleborus species, however, produce nectar continuously for a very long period of time.
bees prefer nectar rich in sucrose. Floral structure suggests that the nectaries of H. foetidus are only accessible to longtongued bees, whereas those of H. bocconei are exposed and
The fact that nectar secretion is not constant in time in H. foetidus may be due to: a. ageing of the secretory cells; b. production is constant over time but nectar loss is proportional to the amount of nectar present; c. nectar production may be inhibited by the presence of nectar in the nectary, as suggested by Galetto and Bernardello (1992 b121l). The concentration in this species increases during exposure, probably due to loss of water, as reported by Schemske (198011), Galetto and Bernardel-
as in most Rannunculaceae (Baker and Baker, 1983 a]1l), and
thus accessible to any type of insect. However, in this species, the lip forces pollinators to "learn" to open the nectary, which insects other than bumble-bees cannot do (Faegri and van der PijI, 19791151).
lo (1992 b]21 I). The nectar is very concentrated from the start of
The quantity of reward offered by these species is particularly high. Each flower may produce a large quantity of sucrose per day. Bumble-bees may visit flowers at low temperatures, but below 5°C the energy cost is two or three times greater than that required at 26°C (Heinrich and Raven, 19721251). Flowers
secretion, and therefore has a high osmotic potential. One would, therefore, expect to find a saturation response: the
pollinated at low temperatures must, therefore, produce a higher calorie reward than those flowering at high tempera-
more concentrated the nectar becomes, the less the water loss. Moreover, the flower, which is less open, offers a more stable environment in terms of humidity.
tures (Heinrich and Raven, 1972125]).
Pollen is generally regarded as a source of protein for pollinators because it is ingested and the cell content is principally digested (Stanley and Linskens, 19741401). Since nectar is generally rich in sugars and without cellular structure, it is mainly regarded as a source of calories. However, bacause the nectar
J. L. Vesprini, M. Nepi. and E. Pacini
568 Plant biol. 1 (1999)
of these two Helleborus species is secreted in a particular way and stains for lipids and proteins, it is more complex than a solution of sugars, and probably a source of other nutrients for pollinators.
Acknowledgements Research
performed under 'Ricerca d'Ateneo", Universit di
Siena, 1999.
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41
E. Pacini Dipartimento di Biologia Ambientale, Sez. Botanica Via P. A. Mattioli 4 53100 Siena Italy E-mail:
[email protected]
Section Editor: G. Gottsberger
