Floral nectary characteristics of five species of Centaurea and Inula (Inula ensifolia, I. salicina, I. spiraeifolia, Centaurea micranthos, C. scabiosa) were examined for using light microscopy and scanning electron microscopy, and the sugar contents were studied in relation to nectary morphology and plant size. The differences in the nectaries, plant size and nectar sugar content of the studied species indicate that the larger plants may reflect a greater allocation to sugar content

Dósa, G.

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Acta Botanica Hungarica 45 (1–2), pp. 65–73, 2003MORPHOLOGY OF NECTARIES IN RELATIONTO NECTAR SECRETION OF INULA AND CENTAUREASPECIES IN THE HUNGARIAN HABITATSG. DÓSASociety of Natural Sciences, H-2100 Gödöllő, Szabadság u. 13, Hungary(Received 13 March, 2002)Floral nectary characteristics of five species of Centaurea and Inula (Inula ensifolia, I. salicina,I. spiraeifolia, Centaurea micranthos, C. scabiosa) were examined for using light microscopy andscanning electron microscopy, and the sugar contents were studied in relation to nectarymorphology and plant size. The differences in the nectaries, plant size and nectar sugar con-tent of the studied species indicate that the larger plants may reflect a greater allocation tosugar content.Key words: floral nectary, glandular tissue, plant size, size and form of nectary, sugar con-tent of nectarINTRODUCTIONAsteraceae is one of the richest-in-species families of the plant kingdom.In spite of its big size we have only a few knowledge about the nectaries of itsspecies, and we can find no articles concerning on the size and the nectar pro-duction of their nectaries.Several articles were born for ultrastructure of nectaries (Fahn 1979), andnectar chemistry (Percival 1961, Baker and Baker 1990, Carlisle and Ryle 1955).The plant species nectar is the principal floral reward (Simpson and Neff1983). Both the quantity and quality of floral nectar vary among species, andthis variation is correlated with pollinator type (Baker and Baker 1975, 1983,Cruden et al. 1983). The characters of the nectar have been correlated withplant characters such as sexual stage, position in an inflorescence, flower age,plant size, and flower size (Rathcke 1992). Nectar production has been shownto be influenced by environmental factors such as time of day (Hodges 1993,Pleasants 1983, Marden 1984), water availability (Pleasants 1983, Zimmerman1983), and humidity (Corbet et al. 1979, Bertsch 1983) and also by developmen-tal factors such as the flower position in the inflorescence (Percival and Mor-gan 1965, Pyke 1978, Devlin et al. 1987), and flower age (Pyke 1978, Schemske1980, Pleasants 1983, Devlin et al. 1987).0236–6495/$ 20.00 © 2003, Akadémiai Kiadó, BudapestThe positions, form and size of nectaries of five Asteraceae species weredetermined, and the correlation between the nectar production (sugar con-tent) and nectary size, nectary structure, and plant size was also studied.MATERIALS AND METHODSStudy sitesThe species were chosen based on their appearance in their natural habi-tats. Inflorescences of the investigated species were collected in July 1997 at thefollowing localities:1. South Hungary: Mecsek Mts, on Tettye Hill, in typical karstic shrub-forest on the southern slope, with forest-steppe and steppe elements such asInula spiraeifolia L.2. North Hungary: in the Aggtelek-karst area, where the natural succes-sion resulted here in semi-dry grassland. Dominant species in this area, e.g.Centaurea scabiosa L., Coronilla varia L., Dorycnium germanicum (Gremli) Rikli,Inula ensifolia L., Inula salicina L., Salvia verticillata L. and Teucrium chamaedrysL., with the strating bordering, rimming, Brachypodium pinnatum (L.) P. B., Ca-rex montana L. and sprouting dicotyledons covering as much as 50% in patch-work.MorphologyFor light microscopic tissue examination the inflorescences were fixed in96% alcohol/glycerine/distilled water (1:1:1), embedded in paraffin, sec-tioned with a microtome, and stained with toluidine blue.For scanning electron microscopy, fresh material and samples were fixedwith glutaraldehyde and postfixed in osmium tetroxide, both buffered withphosphate buffer. Fixed material was dehydrated, critical point-dried, andcoated with a layer of gold. The sections were examined in a scanning electronmicroscope at 10 kV.Nectar collection and nectar sugar analysisThe sampling of nectar was made in July and August 1997. Nectar sam-ples were collected in every hour, repeatedly from the same flowers, from pre-viously isolated inflorescence with tulle. The nectar samples were taken fromrandomly chosen flowers by Whatman no. 1 paper wicks. The temperatureand relative humidity were measured over 12 hours. The nectar analysis was66 G. DÓSAActa Bot. Hung. 45, 2003carried out by thin layer chromatography (TLC), where the application weused 1 l Minicaps. Three kinds of sugars were analysed, sucrose, fructose andglucose (others were not present in the nectar samples). We have used stan-dards. The obtained nectar was unfastened from Whatman paper. The mobilephase was ethyl acetate : ethanol : 60% acetic acid-water : saturated with boricacid (5:2:1:1), the post-derivatization took place with thymol sulphuric reagent(Grösz and Braunsteiner 1989). After the chemical reaction had been takenplace the purple patches were visible on the TLC plates. Area size of patcheswas measured with densitometer. The different sugar concentration wascounted by area size of patches. Baker-scale based on the proportion of threesugars was used for the characterisation of nectar (Baker and Baker 1975,1983).NECTARY MORPHOLOGY AND NECTAR SECRETION OF INULA AND CENTAUREA 67Acta Bot. Hung. 45, 20031 243Figs 1–4. 1: Inula ensifolia: 1 = Surface view of the nectary with stomata (SEM, ×300); 2 = Lon-gitudinal section of the nectary (LM, ×60) (arrow = ripe cells). 3–4: Inula salicina: 3 =Twin-stomata on top of nectary (SEM, ×3,000); 4 = Surface view of nectary (SEM, ×500)RESULTSMorphology of nectariesTheir floral nectary functions as an independent compact unit. They arearound the basal part of the style, like a ring. Their form is truncated. Accord-ing to Swales (1979): “The nectaries of the Asteraceae occur as small swellingsat the base of the style which fit neatly within the narrow tube of the corolla.”The nectaries of the examined species are covered tightly by uniform epider-mis cells, which are rectangle-shaped in medial-longitudal engrave and theyare in one cell layer and they are covered by the thick cuticle (Figs 5–7). Fahn(1979) studied the morphology and ultrastructure of nectaries and he found:“If the cuticle is very thin from inception, …, the nectar permeates through thecuticle. If the cuticle is thick, it may possess pores; …” Nectar is exuded to thesurface via modified stomata (like pores on the nectaries) of gap (Figs 1–8). Theradial-anticlinal cell-wall of the epidermis cells are undulatory a bit and theyare shown as a vertical columned arrangement from outside (Figs 1, 4). Thesubepidermal cells are situated irregularly. The glandular tissue is composedof smaller and isodiametric cells with thin walls, relatively large and stronglypainted nuclei. No intercellulars among the cells . There is a loose parenchymatissue toward the basal of the nectary.According to Pesti (1980) the nectaries of Asteraceae are usually domi-nated of width, however parameters of nectaries cannot simplify according toexaminations. Therefore I constructed categories in order to more exact formand size-determination:I. the quotient of average breadth (l’) and average height (h’) of the nec-taries ( form-determination):a. isodiametric (h’/l’=0.9 – 1.1): breadth and height of the gland are similar.b. anisodiametrici. flattened: h’/l’ 0.9ii. oblong: h’/l’  1.1According to these categories:The nectary-breadth is dominated, anisodiametric and flattened: I. ensi-folia (Figs 1–2), I. salicina (Figs 3–5), I. spiraeifolia (Fig. 6).The nectary-height is dominated, anisodiametric and oblong: C. micran-thos (Fig. 7), C. scabiosa (Fig. 8).II. “Area-multiple”, which is formed about average-height of nectary andaverage-width of nectary in order to complete description ( size-deter-mination).68 G. DÓSAActa Bot. Hung. 45, 2003They have large nectaries (h’· l’  100,000 m2): C. micranthos (Fig. 7), C.scabiosa (Fig. 8). Their glandular tissue can be found until ovary.They have small nectaries, the gland tissue is ring-like and it is separetedfrom neighbouring parts (h’· l’ 100,000 m2): I. ensifolia (Figs 1–2), I. salicina(Figs 4–5), I. spiraeifolia (Fig. 6).NECTARY MORPHOLOGY AND NECTAR SECRETION OF INULA AND CENTAUREA 69Acta Bot. Hung. 45, 20035687Figs 5–8. 5 = Inula salicina. Longitudinal section of nectary (LM, ×60). 6 = Inula spiraeifolia.Longitudinal section of the nectary (LM, ×60). 7 = Centaurea micranthos. Longitudinal sec-tion of the nectary (LM, ×60) (arrow = ripe cells). 8 = Centaurea scabiosa. Lateral view of theupper part of the nectary with stomata (SEM, ×600)The contracted description is following (form and size determination):I. Isodiametric, small = ISIsodiametric, large = ILII. Anisodiametric– flattened, small = AFS (I. ensifolia, I. salicina, I. spiraeifolia)– flattened, large = AFL (C. micranthos)– oblong, small = AOS– oblong, large = AOL (C. scabiosa)There is a cell-row (so-called “ripe cells“) directly under the glandular tis-sue at the species with small nectaries (Fig. 2), which is found in the nectary-parenchyma at the species with large nectaries (Fig. 7). Along the ripe cells atube is separated which includes the stamens, style and nectary. We can findthe ripe cells only in this phase when the inflorescence is ripening.The sugar content of nectar in relation to size of nectaries and plant sizeThe composition of the nectar sugar is examined. Three sugars – themonosaccharides fructose, glucose, the disaccharide sucrose (glucose-fruc-tose) – were encountered in appreciable amounts. Two Centaurea species gavevery few quantifiable fructose and glucose of appreciable amounts. Amongthree sugars relative to the ubiquitous sugar sucrose, and the monosaccha-rides fructose and glucose were not found at every day in the samples, espe-cially at Centaurea. The highest sugar content of nectar is produced at the dateof the peak flower in the early July in Inula species and C. micranthos and thenectar production decreased at the beginning of the blooming period and atthe end, but it does not leave off. C. scabiosa has also been high sugar content atthe date of peak flower but it was not continuously on every day.C. micranthos gives order with the highest content of fructose (31.72%).The highest value was sucrose content/total sugar content in the nectar of C.scabiosa (70.09%). The glucose content takes part in sugar content is the biggestin the nectar of I. spiraeifolia (40.67%) was the highest, the nectar of I. spiraeifoliais hexose-rich (glucose-dominant). The nectar of I. ensifolia and C. micranthosare sucrose-rich, the nectar of I. salicina and C. scabiosa are sucrose-dominantaccording to Baker-scale (S/F+G) (Baker and Baker 1975, 1983, 1990).The difference in nectar sugar content between two genus was as large asthe difference between nectaries size and plants size (Tables 1–2). I found mar-ginally significant positive correlation between the sugar content and plantsize (rS = 0.6242 p  0.0537). The nectary size was correlated with the sugarcontent (rS = 0.7547; p = 0.0116).70 G. DÓSAActa Bot. Hung. 45, 2003DISCUSSIONAccording to Pesti (1980) usually width of nectary dominated to Liguli-florae but Tubuliflorae subfamily showed oblong form e.g. Centaurea species.Poljakov (1967) (Poljakov’s diagram) and later Robinson and Brettell (1973) re-cognised two major (subfamilies) groups in Asteraceae. This family divisioninto two subfamilies neither represents a basal dichotomy nor a sister grouprelationship within the Asteraceae. So, the form of nectary has not been indi-cated as differences between the two subfamilies. The two Centaurea speciesshowed different forms (C. micranthos: flattened, C. scabiosa: oblong).It is worth noting explicitly that nectar concentration and sugar contentvalues appear to be “conservative characters” at least to the genus level, andperhaps in some cases to higher taxonomic levels, as proposed by Percival(1961). Both the quantity and quality of floral nectar vary among species, andthis variation is influenced by environmental factors.According to Baker and Baker (1990) the nectars of Asteraceae are domi-nated by hexose sugars. Sugar content in four species has been shown sucrosedominant and I found one species which is glucose dominant.Although the number of examined species are a few, the correlation mayreflect greater allocation to sugar content by larger plants and larger nectaries.NECTARY MORPHOLOGY AND NECTAR SECRETION OF INULA AND CENTAUREA 71Acta Bot. Hung. 45, 2003Table 1Nectary size of examined speciesNectaryheight (h)[µm]h’[mm]Nectarywidth (l)[mm]l’[mm]h’/l’ h’l’[mm2]Inula ensifolia 110–170 140 230–310 270 0.52 37.800Inula salicina 100–120 110 230–275 250 0.44 27.500Inula spiraeifolia 70–80 75 140–180 160 0.46 12.000Centaurea micranthos 330–460 395 250–380 315 1.25 124.425Centaurea scabiosa 450–560 505 160–400 280 1.8 141.400Table 2Average of the daily total sugar content of examined Inula and Centaurea species, and theBaker-scaleInula Centaureaensifolia salicina spiraeifolia micranthos scabiosaSugar content (mg/ml) 18.5008 22.1348 9.2103 58.6527 34.2068Baker-scale (S/(F+G)) 0.9915 1.41 0.4253 0.9075 2.3443*Acknowledgement – The main part of this study was undertaken at the Janus PannoniusUniversity, Department of Botany where I worked in the laboratory.REFERENCESBaker, H. G. and Baker, I. (1975): Nectar constitution and pollinator-plant coevolution. – In:Gilbert, L. E. and Raven, P. H. (eds): Animal and plant coevolution. Texas UniversityPress, Austin, pp. 100–140.Baker, H. G. and Baker, I. (1983): Floral nectar constituents in relation to pollinator type. – In:Jones, C. E. and Little, R. J. (eds): Handbook of experimental pollination biology. VanNostrand-Reinhold, New York, pp. 117–141.Baker, H. G. and Baker, I. (1990): The predictive value of nectar chemistry to the recognitionof pollinator types. – Isr. J. Bot. 39: 157–166.Bertsch, A. (1983): Nectar production of Epilobium angustifolium L. at different air humid-ities; nectar sugar in individual flowers and the optimal foraging theory. – Oecologia59: 40–48.Carlisle, E. and Ryle, M. (1955): The influence of nitrogen, phosphate, potash and lime onthe secretion of nectar by red clover in the field. – Empire I. Exp. Agric. 23: 126–130.Corbet, S. A., Unwin, D. M. and Prys-Jones, O. E. (1979): Humidity, nectar, and insect visitsto flowers, with special reference to Crataegus, Tilia and Echium. – Ecol. Entom. 4:9–22.Cruden, R. W., Hermann-Parker, S. M. and Peterson, S. (1983): Patterns of nectar productionand plant-pollinator coevolution. – In: Elias, T. S. and Bentley, B. A. (eds): Biology ofnectaries. Columbia University Press, New York, USA, pp. 80–125.Devlin, B., Horton, J. B. and Stephenson, A. G. (1987): Patterns of nectar production ofLobelia cardinalis. – Amer. Midland Nat. 117: 289–295.Fahn, A. (1979): Ultrastructure of nectaries in relation to nectar secretion. – Amer. J. Bot.66(8): 977–985.Grösz, J. and Braunsteiner, W. (1989): Quantitative determination of glucose, fructose andsucrose, and separation of fructo-oligosaccharides by means of TLC. – J. Planar Chro-matography 2: 420–423.Hodges, S. A. (1993): Consistent interplant variation in nectar characteristics of Mirabilismultiflora. – Ecology 74: 542–548.Marden, J. H. (1984): Intrapopulation variation in nectar secretion in Impatiens capensis. –Oecologia 63: 418–422.Percival, M. S. (1961): Types of nectar in Angiosperms. – New Phytol. 60: 235–281.Percival, M. S. and Morgan, P. (1965): Observations on the floral biology of Digitalis species.– New Phytol. 64: 1–22.Pesti, J. (1980): Connection of structure and production in floral nectaries of Compositae. – PhDthesis, Körmend, Hungary.Pleasants, J. M. (1983): Nectar production patterns in Ipomopsis aggregata (Polemonia-ceae). – Amer. J. Bot. 70: 1468–1475.Poljakov, P. P. (1967): Systematics and origin of the Compositae. – Nauka, Alma-Ata.Pyke, G. H. (1978): Optimal foraging in bumblebees and coevolution with their plants. –Oecologia 36: 281–293.72 G. DÓSAActa Bot. Hung. 45, 2003Rathcke, B. J. (1992): Nectar distributions, pollinator behavior, and plant reproductive success. –In: Hunter, M. D., Ohguishi, T. and Price, P. W. (eds): Effects of resource distributionon animal-plant interactions. Academic Press, New York, USA, pp. 113–138.Robinson, H. and Brettell, R. D. (1973): Tribal revisions in Asteraceae. VIII. A new tribe,Ursinieae. – Phytologia 26: 76–86.Schemske, D. W. 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Morphology of nectaries in relation to nectar secretion of Inula and Centaurea species in the Hungarian habitats

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