560 Original Paper

Nectary Structure, Nectar Secretion Patterns andNectar Composition in Two Helleborus Species

J. L. Vesprini1'2, M. Nepi2, and E. Pacini2

Facultad de Ciencias Agrarias, UNR CC14 2123 Zvalla, Argentina2 Dipdrtimento di Biologia Ambientale, Università di Siena, Italy

Received: April 9, 1999; Accepted: July 10, 1999

Abstract: The morphological and cytological characteristics ofnectaries of Helleborus foetidus and H. bocconei during the secre-tory period are reported. The nectaries are derived from modi-fied petals and secrete nectar continuously for about 20 days;they consist of a single layered epidermis, nectar-producing par-enchyma and photosynthesizing parenchyma. Nectar secretionis holocrine and the nectar is released by rupture of the walland cuticle of each epidermal cell. The nectaries of the two spe-cies differ in number and external morphology. In H. foetidus, se-cretion begins before anthesis and secretion rate decreases withnectary age. In H. bocconei it begins on the day of anthesis andproceeds at a constant rate. The nectar has a high sugar content,mainly sucrose, and also contains lipids and proteins.

Key words: Nectary structure, secretion mechanisms, nectarsecretion patterns, nectar sugar composition, Helleborus.

Introduction

The nectary is a common secretory tissue in angiosperms; itproduces nectar, a sugar solution which is regardedas one ofthe main rewards for pollinators in many species (Fahn,1979116]). Nectaries may have different morphologies, may besituated in vegetative (extrafloral nectaries) or reproductiveparts of plants (floral nectaries) and may have different ana-tomical origins (Fahn, 1979[6]). This is why the term nectarydoes not refer to a single, well defined anatomical structure.Nectaries usually consist of an epidermis and a parenchymaspecialized for nectar production (Fahn, j979116]; Durkee,19821101). The cells of this tissue are generally small, with dense

cytoplasm and a relatively large nucleus (Durkee, 1982110]). Theparenchyma may perform photosynthesis, providing materialsfor nectar production, or it may store starch from other photo-synthesizing parts of the plant (Nepi et al., 19961311 and refer-ences therein).

Nectar may emerge from the nectary in different ways, some ofwhich are related to nectary anatomy. The most common ofthese are: 1) through stomata in the epidermis: 2) through a

thin permeable cuticle; 3) through pores in the cuticle; 4) afterrupture of the cuticle under nectar pressure. Secretion is gen-erally eccrine or granulocrine (Fahn, 19881181).

Nectar may be regarded as an aqueous solution of sugars,mainly sucrose, glucose and fructose. Amino acids and pro-teins (mostly enzymes) are other common constituents. Var-ious substances that vary from species to species may be pres-ent in traces (Baker and Baker, 1983 a]1]).

Nectar composition and its secretory pattern vary betweenspecies and within the same species in relation to environ-mental parameters (Fahn, 19791161; Cruden et al., 198316]: Mar-den, 19841301; Freeman and Head, 19901191; Wyatt et al.,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 tothe sexual phases (Gillepsie and Henwood, 19941221). It was re-cently shown that the flower itself can modify nectar composi-tion, even after its secretion, by reabsorption of sugars (Crudenet al., 198316]; Burquez and Corbet, 199115]; Nicolson, 1995132];Nepi et al., 19961311: Davis, 1997; Koopowitz and Marchant,1998129]).

The present study is concerned with nectary structure andnectar secretion and composition in Helleborus foetidus andHellebonis bocconei, two species which flower synchronouslyin winter and are pollinated by bumble-bees (personal obser-vations). Since these plants are not apomictic and automaticpollination does not occur, they depend on pollinators for seedproduction.

Herrera and Soriguer (1983126]) studied inter- and intrafloralvariation in nectar production in populations of H. foetidusgrowing in southern Spain and found wide variations in thequantity of nectar in flowers at the same stage of anthesis.Eymé (1966113]) and Eymé and Le Blanc (19631141) studied theultrastructure of the nectary of H. foetidus. Other authors havereported 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 severalplants, including two Hellebonis species.

Plant biol. 1(1999) 560-568© Georg Thieme Verlag Stuttgart. New YorkISSN 1435-8603

Nectar and Nectaries in the Genus Helleborus Plant biol. 1 (1999) 561

Materials and Methods

Study area

Populations of H. bocconei and H. foetidus growing in a decid-uous submediterranean Quercus cerris forest near Monterig-gioni (Central Italy, Province of Siena, 43°23'38"N, 11 °40'18"E,elevation 213 m) were studied in the field and sampled duringJanuary, February and March of 1997 and 1998.

Nectary structure

Samples were obtained from flowers of both species in fourstages: I) before opening of the flower; 2) on the first day ofanthesis; 3)10 days after the beginning of anthesis; 4) beforethe nectaries fell, i.e., 20 days after flower opening. Flowers ofboth species are protogynous. Since the phases of anthesis var-ied considerably in length, flowers in stages 3 and 4 belongedto different phases: hermaphrodite, male and beyond the peri-od of anthesis.

Samples were fixed in 5% glutaraldehyde in phosphate buffer(pH 6.9), dehydrated in an ethanol series and embedded inTechnovit 7100 (Heraeus Kulzer GmbH).

Semithin sections were stained with: a) toluidine blue (TBO)(O'Brien and McCully, 19811331) as general stain; b)PAS for totalinsoluble polysaccharides (O'Brien and McCully, 1981133]); c)auramine 0 for cuticle (Heslop-Harrison, 1977127]); d) calco-fluor for cellulose (Hughes and McCully, 1975128]); e) FITC forproteins (Pearse, 1968]]).

To observe photosynthesis by nectaries, thin hand-cut sections(20—50 m thick) were examined by fluorescence microscopyfor autofluorescence of chioroplasts.

Pattern of nectar secretion

Before anthesis commenced, 40 flowers of H. bocconei and 36of H. foetidus were bagged with voile bags to exclude pollina-tors. Different treatments were randomly assigned to eachflower, the treatment consisted of the removal of nectar at dif-ferent times after flower opening. Each flower was sampledonly once at a fixed age by removal of nectar. Where youngflowers were sampled (up to 15 days old), these flowers wereresampled 10 days later.

The nectar was collected with graduated micropipettes fromall nectaries of each flower. The volume of nectar from eachflower was recorded and the sugar concentrations measuredby means of a refractometer (0—90 Brix). These two param-eters enabled us to calculate sugar production, as describedby Bolten et al. (1979]]).

In H. foetidus, the nectar was withdrawn at the followingtimes: one day before opening of the flower, the day afteropening, and 5, 10, 15 and 20 days after opening. Six flowerswere sampled at each time. On day 20, part of the nectar wasfound on the tepals, and since it could not be collected, thedata for this time point were excluded from the statisticalanalysis for this species.

Fig.1 Flower and nectary structure in both species. (A) Section of aflower of H. foetidus x 1 (nectaries arrowed). (B) Front and side viewsof a nectary of H. foetidus x 3. (C) Section of a flower of H. bocconeix 0.8 showing nectaries (arrows). (D) Front and side views of a nec-tary of H. bocconei x 3.

InH. bocconei the nectar was withdrawn: one day before open-ing of the flower, the day of opening, and 5,8,11,14,17 and 20days after opening. Five flowers were sampled at each time.

Statistical analysis

The variables volume, concentration and total sugars had anormal distribution and were therefore processed by regres-sion analysis using the programme Statistica for Windows(Statsoft, 1993141]).

Nectar composition

In both species, sampling for the determination of sugars wascarried out during the following floral phases: female phase,hermaphrodite phase, male phase. Nectar samples were storedat — 80°C until analysis. Sucrose, D-glucose and D-fructoseconcentrations were determined with Boehringer Mannheimkit no. 716260 based on spectrophotometric determination ofenzyme reduction of NADP at 340 nm. Before measurement,the samples were diluted 1: 300 with distilled water. Standardsolutions of carbohydrates were measured as controls. The re-sults were expressed in mg/ml of nectar.

Lipids and proteins were identified by spot staining methodsas described in Dafni (199218]).

Results

Nectary morphology

The nectaries are inside a corona of five green tepals. In H.foe-tidus, the flower is bowl-shaped and the nectaries are not di-rectly exposed (Fig. 1 A). In H. bocconei, the flower is cup-shaped and the nectaries are more exposed (Fig. I C). In bothspecies, the nectaries are green and show the typical red fluo-

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562 Plant biol. 1 (1999) J. L. Vesprini, M. Nepi, and E. Pacini

Fig.2

Nectar and Nectaries in the Genus He!Ieborus Plant biol. 1 (1999) 563

rescence of chlorophyll when observed by microscope underUV light. They are attached to the receptacle by a stalk whichis more evident in H. bocconei and shorter in H. foetidus(Fig.1). In the latter, the nectaries are tubular (5 x 1.5 mm) andthe distal part has a toothed border and is open (Fig. 1 B). In H.bocconei the nectaries are more or less conical with an expand-ed distal part (3 mm) having a lip closing the nectary orifice(Fig. I D). In both species, the nectar-producing parenchyma isin the proximal area. The mean number of nectaries was 10.45per 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

The nectary structure is characterized by an external photo-synthesizing parenchyma, a nectar-producing parenchymaarid an internal epidermis (Fig. 2A). The nectar-producing par-enchyma consists of small, more or less isodiametric, poly-hedral cells without intercellular spaces (Fig. 2). The cell wallscontain small amounts of cellulose and are situated in the prox-imal part of the tubular nectary between the vascular bundlesfrom the stalk (Fig. 2A). The parenchyma tapers distally, sothat it is V-shaped. The red autofluorescence of chlorophyll isonly observed in the outer cell layers of the parenchyma.

The internal epidermis covering the nectar producing paren-chyma consists of a single layer and is without stomata. Instage lit consists of uniform cylindrical cells, the longitudinalaxes of which are disposed perpendicular to the nectary sur-face 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 superficiallayers, 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 cellsincreases and their morphology is more heterogeneous untilstage 4, when the epidermis of the proximal part consists of

Fig.2 Nectary of H. foetidus. (A) Stage 2, magnification x60, PAS.The inner epidermis (ie) surrounding the nectar cavity (nc) consistsof a single layer and the cells have their major axis perpendicular tothe nectary surface in the basal part. The nectar-producing paren-chyma (np) is situated between the vascular bundles (vb) and the in-ternal epidermis (ie), and consists of small cells. Photosynthesizingparenchyma (pp) is located between external epidermis (ee) and vas-cular bundles (vb). (B) Stage 2. magnification x 150, PAS. As secre-tion proceeds, overlapping cells appear in the epidermis (arrow). Inthe parenchyma, especially in the more superficial layers, cells are ar-ranged in a regular pattern of groups of four. This organization is lostdeeper in the tissue. The wall of the secreting cell bulges into thenectary cavity (arrowhead). (C) Stage 2, magnification x 65, auramine0. The cuticle covering the epidermis is continuous. (0) Stage 4.magnification x 175, TBO. The internal epidermis (ie) in the basalarea has lost its single-layered structure and the cells have hetero-geneous morphology. (E) Stage 3, magnification x 230, TBO. The oc-casional empty cell which has already secreted nectar is visible in theepidermis. The space it leaves has the form of an hour-glass due toencroachment by adjacent cells. (F) Stage 2, magnification x 300,PAS. Secretory activity begins with bulging of the wall protrudinginto the nectary cavity. (G) Stage 2, magnification x 300, PAS. Thewall then ruptures and the secretion builds up under the cuticle. (H)Stage 2, magnification x 300. PAS. The cuticle breaks and the secre-tory material is released.

two layers and the cells may be isodiametric, cylindrical or fu-siform (Fig. 2D). In stage 3, a few empty cells shaped like hour-glasses are observed in the epidermis, next to cells with convexwalls (Fig. 2E). At this stage, the regular organization of thegroups of four cells is lost in the parenchyma underlying theepidermis. In stage 2, 3 and 4, the wall and overlying cuticleof the secretory cells contain breaks (Fig.2H).

Nectar secretion in H. foetidus

Nectar secretion occurs via the inner epidermis and secretoryactivity is not simultaneous in all epidermal cells, but occurs insingle cells in an apparently random fashion. In stage 1, secre-tory cells are present in the proximal part, but as secretory ac-tivity proceeds (stages 3 and 4), secreting cells appear in moredistal positions.

The walls of secretory cells bulge slightly into the nectary cav-ity 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 aprotrusion (Fig. 2G) which grows until it bursts (Fig. 2H). Thewall and the cuticle then collapse, covering the space pre-viously occupied by the secreting cell (Fig. 2E). The secretorymaterial 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 ismuch stronger than in H.foetidus. The epidermis covering theparenchyma in stage 1 consists of a single layer and is moredisorganized in the proximal part in later stages (Fig. 3B).Thecuticle overlying the epidermis is uninterrupted in stage I anddevelops breaks in stages 2 (Fig.3C), 3 and 4. The secretorycells become club-shaped and protrude into the nectary cavity(Fig. 3D); theirwalls appear stretched and lacerated in PAS andTBO stained sections (Fig. 3D).

Except in the layers close to the internal epidermis, the nectar-producing parenchyma and the photosynthesizing parenchy-ma contain amyloplasts which are most abundant in stage 2and rare in stages I and 4 (Fig. 3A).

Secretion in H. bocconei

The secretory material emerges due to rupture of the individ-ual cell walls (Fig. 3E) and then the cuticle (Fig. 3C), which hasinterruptions where secretion has occurred. The secretory ma-terial is weakly positive to TBO, FITC and PAS. The spaces re-maining between epidermal cells after secretory activity hasoccurred are smaller than in H. foetidus because the secretorycells protrude into the nectary cavity (Fig.3B). The temporalsequence of activity of the secretory cells is similar to that ob-served 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 nec-taries fall. The longer the flowers were left before the first col-lection of nectar, the greater were the volume and concentra-tion, and hence sugar content (Fig. 4). The total volume of nec-

564 Plant biol. 1 (1999) J. L. Vesprini, M. Nepi, and E. Pacini

Fig. 3 The nectary of H. bocconei. (A) Stage 2, magnification x 40,PAS. The nectar-producing parenchyma (np) is visible under the epi-dermis (ie) between vascular bundles (vb). Amyloplasts are visibledeep in the parenchyma and in the outermost cells at the base ofthe nectary. (B) Stage 3, magnification x 130, TBO. The epidermis inthe basal area is no longer single layered, the cells are irregular andthere are empty intercellular spaces (asterisk). (C) Stage 2, magnifi-

tar,sugar concentration and rate of sugar production showed anegative 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 decreasedwith increasing nectary age (Fig. 5C).

cation x 75, auramine 0. The cuticle of bulging cells is thinner (ar-row) and is broken where secretion has occurred (arrowhead). (D)Stage 2, magnification x 200, PAS. The secreting cells are club-shaped and protrude into the nectary cavity (nc); the walls are lacer-ated. (E) Stage 4, magnification x 200, TB0. Secretion occurs by rup-ture of cell wall and cuticle.

In H. bocconei, nectar secretion begins when the flower opens(Fig. 6) and lasts about 20 days. Again, the longer the flowerswere left before nectar collection, the greater were the volumeand concentration, and hence sugar content (Fig. 6). Volumeand concentration showed a quadratic pattern in relation totime, negative in the case of volume and positive for concen-tration (Figs. 6A,B). Hence, the rate of sugar production had asecond order regression with a linear principal component(Fig. 6C). This means that in nectaries of different ages that

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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. Sincepart of the nectar was found on the tepals on day 20, the data atthis time point were exduded from the analysis. (A) The volume ofnectar secreted increased with the age of the nectary. However theincrease had a negative quadratic component. The rate of nectar se-cretion, therefore, decreased with the age of the nectary. (B) Nectarconcentration showed the same pattern as volume. The increasemay be due to water loss. (C) Sugar production, therefore, had thesame pattern as concentration and volume. The quantity of sugarsproduced increased with age of the nectary, but rate of secretionwas inversely related to age of nectary.

have secreted nectar for the same period of time (10 days),sugar content is almost the same (Fig. 7C).Similarly, concen-tration and volume did not show any significant regression(Figs. 7A,R).

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Fig. 5 Nectar secretion over a 10-day period in H. foetidus nectariesof different age. (A) Volume decreased with time; the rate of de-crease was positive quadratic. Older nectaries secreted less nectarthan young ones in the same interval of time. (B) Concentration didnot vary with age of nectary, indicating that water loss is proportion-al to exposure time. (C) Sugar production, therefore, decreased withage of the nectary.

Nectar composition

Sucrose was the main nectar sugar in these species and nectarcomposition did not change in the different phases of the lifeof the flower. Fructose and glucose constituted less than 5% ofthe sugar, and were completely absent in some samples. Meansucrose concentrations were high in both species, being607.81 mg/mI in H.foetidus and 275.18 mg/mI in H. bocconei.

The nectar of both species stained positively for proteins andlipids.

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Fig. 7 Nectar secretion over a 10-day period in H. bocconei nectariesof different ages. (A) Volume decreased slightly in older nectaries(not statistically significant). (B) Concentration did not vary with ageof nectary, indicating that water loss was proportional to exposuretime. (C) Sugar production, therefore, did not decrease with age ofnectary, confirming that production is constant in time.

taries ofSambucus nigra, secretion is not holocrine but followsthe merocrine model, with the cells dying at the end of thesecretory process.

Discussion

Nectar secretion

The manner of secretion of the floral nectaries ofthe two spe-cies of Helleborus is unusual. Nectar secreting cells normallyremain integral throughout the period of secretion and secre-tion is merocrine (Fahn, 1988118]). Only in rare cases, all ofwhich have been in extrafloral nectaries, has lysogenic secre-tion been reported (Fahn, 1987117] and references therein).However, Fahn (19871171) underlined that in the extrafloral nec-

If secretion involving emptying of epidermal cells is postulat-ed, one would expect to find an epidermis with many intercel-lular spaces and a cuticle with many breaks in stages 3 and 4.However, few intercellular spaces were observed and the cu-ticle was more or less intact. It therefore seems likely that theloss of cells derived from this mode of secretion is compensat-ed by reorganization of the cells of the nectar-producing tis-sue, facilitated by the relative lack of cellulose in the walls,and by repair of the cuticle. Self-repair of this type is necessaryin view of the long period of activity of the nectary.

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Nectar and Nectaries in the Genus Helieborus Plant biol. 1 (1999) 567

It is not clear how the empty spaces are filled. Two hypothesesare: i. that adjacent cells gain space by occupying the emptyspaces (Fig. 3D); ii. that the inferior cell is pushed by underly-ing ones into the space left by the empty cell (Fig. 2 E). The sec-ond, 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 substancesphotosynthesized by the nectary itself or by other parts of theflower or plant (Fahn, 19791161). An abundance of reserve sub-stances is generally stored when a large amount of nectar isproduced in a short time (Nepi et al., 1996131]). In extrafloralnectaries, which generally produce nectar for longer periodsthan floral ones, starch is not normally stored and the nectariesoften perform photosynthesis (Baker et al., 197813]; Durkee,1982110]; Eleftheriou and Hall, 19831111; Fahn, 19871171; Groutand Williams, 19801241; Galetto and Bernardello, 1992 a1201; Vi-noth and Yash, 19921421). The floral nectaries of the two Helle-borus species can be regarded as "slow producers" accordingto the classes of Cruden et al. (198316]). They perform photo-synthesis, which suggests that at least part of the nectar is pro-duced "in situ". Our data does not allow us to estimate howmuch nectar is produced by the nectary and how much byphotosyrithesizing parts. However it seems likely that the nec-tary contribution would be the larger in H. bocconei because:1) the flower is more open and the nectaries more exposed tolight; 2) nectar is offered as soon as the flower opens, that is,when the nectaries are exposed; 3) autofluorescence of chloro-phyllis more intense than in H. foetidus; 4) sugar productionis constant over time; 5) small quantities of starch were ob-served in the cells of more superficial parts of the nectary, clos-er to vascular bundles.

Pattern of nectar secretion

The duration of floral anthesis varies according to species (Pri-mack, 198511). In the case of long-lived flowers, the nectariesmay produce nectar over a limited period, the nectar remainsin the flower until it is taken by pollinators; but may wait for along time, as in certain orchids (Endress, 19941121). The nectar-ies of our Helleborus species, however, produce nectar continu-ously for a very long period of time.

The fact that nectar secretion is not constant in time in H. foe-tidus may be due to: a. ageing of the secretory cells; b. produc-tion is constant over time but nectar loss is proportional to theamount of nectar present; c. nectar production may be inhib-ited by the presence of nectar in the nectary, as suggested byGaletto and Bernardello (1992 b121l). The concentration in thisspecies increases during exposure, probably due to loss of wa-ter, as reported by Schemske (198011), Galetto and Bernardel-lo (1992 b]21 I). The nectar is very concentrated from the start ofsecretion, and therefore has a high osmotic potential. Onewould, therefore, expect to find a saturation response: themore concentrated the nectar becomes, the less the water loss.Moreover, the flower, which is less open, offers a more stableenvironment in terms of humidity.

Nectar losses on day 20 were due to the position of the flower,which faces the ground, and to nectary structure. In this spe-cies, the nectary is open, so that when nectar volume is consid-erable, it may drip out of the nectary.

In H. bocconei, the concentration and volume of nectar secret-ed followed the opposite pattern. Again, evaporation caused apositive increase in concentration and determines a negativeincrease in the volume over time. The increase in concentra-tion was different from that in H. foetidus. The reasons may bethat the nectar is more dilute than that of H.foetidus, especiallyduring the first days of secretion, and it therefore loses watermore easily. The flower of this species is more open and hencethe nectar is more subject to the effects of the environment,despite the lip protecting the nectary opening. The effects offlower morphology in determining microclimatic protectionof the nectar is demonstrated by Corbet et al. (197917]) in H.foe-tidus and H. lividus Aiton ssp. corsicus (Willd.) Tutin and otherplant species.

Herrera and Soriguer (1983126]) report that, in populations ofH. foetidus growing in southern Spain, the nectaries secretednectar from the beginning of the female phase until half waythrough the male phase, when they fell. Since the duration ofanthesis is variable (10—26 days) and temperature-dependent,nectar secretion may cease before anthesis ends or may con-tinue beyond the period of pollen exposure. In fact, we some-times observed flowers which had lost their anthers and wereforming fruit, but which had active nectaries still visited bypollinators.

Pollinators and nectar composition

Sugarcomposition, nectar volume (Baker and Baker, 1983 b121)and flower structure (i.e., Faegri and van der Pijl, 1979115]) maybe related to the type of pollinator that visits the flower. Thenectar of H.foetidus and H. bocconei consists largely of sucrose,as in most Rannunculaceae (Baker and Baker, 1983 a]1l), andour data are in accordance with the finding of Corbet et al.(1979171). The flowers are visited predominantly by Bombusterrestris and other species of bumble-bees. Baker and Baker(1983b12]) report that long-tongued insects such as bumble-bees prefer nectar rich in sucrose. Floral structure suggeststhat the nectaries of H. foetidus are only accessible to long-tongued bees, whereas those of H. bocconei are exposed andthus accessible to any type of insect. However, in this species,the lip forces pollinators to "learn" to open the nectary, whichinsects other than bumble-bees cannot do (Faegri and van derPijI, 19791151).

The quantity of reward offered by these species is particularlyhigh. Each flower may produce a large quantity of sucrose perday. Bumble-bees may visit flowers at low temperatures, butbelow 5°C the energy cost is two or three times greater thanthat required at 26°C (Heinrich and Raven, 19721251). Flowerspollinated at low temperatures must, therefore, produce ahigher calorie reward than those flowering at high tempera-tures (Heinrich and Raven, 1972125]).

Pollen is generally regarded as a source of protein for polli-nators because it is ingested and the cell content is principallydigested (Stanley and Linskens, 19741401). Since nectar is gener-ally rich in sugars and without cellular structure, it is mainlyregarded as a source of calories. However, bacause the nectar

568 Plant biol. 1 (1999) J. L. Vesprini, M. Nepi. and E. Pacini

of these two Helleborus species is secreted in a particular wayand stains for lipids and proteins, it is more complex than asolution of sugars, and probably a source of other nutrientsfor pollinators.

Acknowledgements

Research performed under 'Ricerca d'Ateneo", Universit diSiena, 1999.

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E. Pacini

Dipartimento di Biologia Ambientale, Sez. BotanicaVia P. A. Mattioli 453100 SienaItalyE-mail: pacini@unisi.it

Section Editor: G. Gottsberger