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. 2004 Jun 30;94(2):269–280. doi: 10.1093/aob/mch137

Floral Nectaries, Nectar Production Dynamics and Chemical Composition in Six Ipomoea Species (Convolvulaceae) in Relation to Pollinators

LEONARDO GALETTO 1, GABRIEL BERNARDELLO 1,*
PMCID: PMC4242162  PMID: 15229123

Abstract

• Background and Aims Floral nectaries and nectar features were compared between six Argentinian Ipomoea species with differences in their pollinator guilds: I. alba, I. rubriflora, I. cairica, I. hieronymi var. hieronymi, I. indica, and I. purpurea.

• Methods Pollinators were recorded in natural populations. The morpho-anatomical study was carried out through scanning electron and light microscopy. Nectar sugars were identified via gas chromatography. Nectar production and the effect of its removal on total nectar sugar amount were determined by using sets of bagged flowers.

• Key Results Hymenopterans were visitors of most species, while hummingbirds visited I. rubriflora and sphingids I. alba. All the species had a vascularized discoidal nectary surrounding the ovary base with numerous open stomata with a species-specific distribution. All nectar samples contained amino acids and sugars. Most species had sucrose-dominant nectars. Flowers lasted a few hours. Mean nectar sugar concentration throughout the lifetime of the flower ranged from 34·28 to 39·42 %, except for I. cairica (49·25 %) and I. rubriflora (25·18 %). Ipomoea alba had the highest nectar volume secreted per flower (50·12 µL), while in the other taxa it ranged from 2·42 to 12·00 µL. Nectar secretion began as soon as the flowers opened and lasted for a few hours (in I. purpurea, I. rubriflora) or it was continuous during the lifetime of the flower (in the remaining species). There was an increase of total sugar production after removals in I. cairica, I. indica and I. purpurea, whereas in I. alba and I. rubriflora removals had no effect, and in I. hieronymi there was a decrease in total sugar production.

• Conclusions The chemical composition, production dynamics and removal effects of nectar could not be related to the pollinator guild of these species. Flower length was correlated with nectary size and total volume of nectar secreted, suggesting that structural constraints may play a major role in the determination of nectar traits of these species.

Key words: Nectary structure, nectar chemical composition, nectar production dynamics, nectar removal effects, pollinators, Ipomoea, Convolvulaceae, morning glory, Argentina

INTRODUCTION

Floral nectar is widely known as the key reward offered by animal-pollinated plants to their pollen vectors (Proctor et al., 1996). This exudate is secreted by nectaries, i.e. glandular tissues located on various floral parts whose features are significant in plant taxonomy and phylogeny (Fahn, 1979). Sugars dominate the total solutes in floral nectar: these are mainly sucrose, fructose and glucose in varying proportions according to the species (Baker and Baker, 1983a, b; Freeman et al., 1991; Stiles and Freeman, 1993). Other compounds, such as amino acids, phenols, lipids and antioxidants, are found as well, but mostly in trace quantities (Baker and Baker, 1975, 1983a). All these substances often impart a particular taste and odour that may be essential for maintaining certain pollinator groups (Southwick, 1990). In many cases it has been interpreted that pollinators determine nectar components and, thus, the nectar sugar ratios together with flower and inflorescence morphology may be good predictors of the pollinators (cf. Baker and Baker, 1990). For instance, hummingbird- and hawk-moth-pollinated flowers tend to produce sucrose-dominant nectar, whereas bee-pollinated flowers tend to produce nectars with a predominance of hexose (Baker and Baker, 1983a, b). In addition, experimental studies on sugar preferences of hummingbirds have demonstrated that they preferred sucrose solutions instead of equivalent monosaccharide ones (e.g. Martínez del Río, 1990; Stromberg and Johnsen, 1990). However, in other instances nectar composition may be a conservative character due to phylogenetic constraints (cf. Galetto et al., 1998).

Nectar is secreted with particular rhythms, throughout the lifespan of a flower, which allow the nectar production dynamics of a species to be determined. Knowledge of nectar production dynamics is fundamental to the understanding of the plant–animal relationship; aspects such as the plant's strategy of offering nectar, the activity patterns, frequency and diversity of pollinators of a plant species, the rates of nectar consumption by animals, among others, could not be understood without it. Nectar production may show diverse patterns according to the different guilds of pollinators that visit the flowers (e.g. Feinsinger, 1978; Cruden et al., 1983; Galetto and Bernardello, 1992), leading to the assumption that there are coevolutionary relationships between nectar traits and pollinator type (Baker and Baker, 1983a, b). For instance, hawk-moth-pollinated flowers produce abundant nectar with low concentration values and bee-pollinated flowers secrete compartively less nectar with higher concentrations, whereas hummingbird-pollinated flowers show intermediate values (e.g. Pyke and Waser, 1981; Opler, 1983; Baker and Baker, 1983a; Sutherland and Vickery, 1993).

At the same time, the effect of nectar removal by floral visitors may have a pronounced effect on the total amount secreted by a flower. Although in some species removal does not modify nectar production (e.g. Galetto and Bernardello, 1993, 1995; Galetto et al., 2000), in others the total amount of sugar in the nectar may be either increased (e.g. Pyke, 1991; Galetto and Bernardello, 1995; Castellanos et al., 2002) or decreased (e.g. Galetto and Bernardello, 1992; Bernardello et al., 1994; Galetto et al., 1997). Predictions for these patterns are not straightforward because they may be related to pollinators, environmental factors, plant resource allocation, or other factors.

Six sympatrically occurring Ipomoea species that have differences in the pollinator guilds, floral colours and breeding systems were chosen to examine their floral nectaries, nectar components and nectar production dynamics to evaluate if there are correlations among these features and to consider the results in the context of plant–pollinator interactions. Ipomoea (whose species are commonly known as ‘morning glory’) is a cosmopolitan climbing genus from warm and pantropical regions with approx. 650 species (Austin and Huáman, 1996) with large showy flowers that are easy to manipulate. Its members have trumpet-shaped flowers of different colours—mainly white, purple, blue, pink, red (Cronquist, 1981). These are visited by a diverse array of animals, including bees, hawk moths, beetles, butterflies, long-tongued flies, hummingbirds and bats (e.g. van der Pijl, 1954; Vogel, 1954; Schlising, 1970; Sobreira-Machado and Sazima, 1987; McDonald, 1991). These visitors look for the floral nectar secreted by a discoidal nectary surrounding the ovary base (Fahn, 1979; Cronquist, 1981). In addition, extrafloral nectar and nectaries are widespread in Ipomoea in petioles and/or in epals that are mostly visited by ants and serve as a herbivore defence mechanism (Elias, 1983; Keeler and Kaul, 1984).

In spite of the attractiveness of the flowers of this diverse genus and the importance of some species as crops or invas-ives (Austin and Huáman, 1996), studies on the floral nectar features of the genus are few. Only five taxa have been examined for their floral nectar composition (Keeler, 1977, 1980; Stucky and Beckmann, 1982; Freeman et al., 1985, 1991), and only six species have been incidentally examined for their nectar secretion (Real, 1981; Stucky and Beckmann, 1982; Stucky, 1984; Devall and Thien, 1989). The present work was undertaken to study and compare the floral nectaries and nectar features in six Argentinian Ipomoea species addressing the following questions: (1) What are the local flower animal visitors? (2) What is the floral nectary structure? (3) What is the chemical composition of the nectar? (4) What are the production dynamics of nectar throughout the lifetime of the flower? (5) What is the floral response to nectar removal? We expected to find differences in nectar sugar composition and production dynamics among the species as they are visited by different pollinator guilds (see above).

The species studied included: I. alba (subgen. Quamoclit) with long, white, hawk-moth-pollinated flowers (McDonald, 1991), I. rubriflora (subgen. Quamoclit) with medium-sized, red, allegedly hummingbird-pollinated flowers (cf. Wilson, 1960; Austin, 1975) and I. cairica (subgen. Quamoclit) with violet-pink flowers, I. hieronymi var. hieronymi (subgen. Eriospermum) with pink flowers, I. indica (subgen. Ipomoea) with blue flowers and I. purpurea (subgen. Ipomoea) with pink, white, or purple flowers, all bee-pollinated (Real, 1981; Maimoni-Rodella et al., 1982; Maimoni-Rodella and Rodella, 1992; Pinheiro and Schlindwein, 1998; Galetto et al., 2002). Ipomoea rubriflora and I. hieronymi are endemic to Bolivia and Argentina (Chiarini and Ariza Espinar, 2004), whereas the other species are mainly pantropical (Austin and Huáman, 1996; Chiarini and Ariza Espinar, 2004). Regarding their breeding system, some species are self-compatible (SC), such as I. alba (Martin, 1970), I. purpurea (Chang and Rausher, 1999; Galetto et al., 2002), and I. rubriflora (L. Galetto, unpubl. res.), whereas others are self-incompatible (SI), such as I. hieronymi (L. Galetto, unpubl. res.), I. indica (Martin, 1970), and I. cairica (Maimoni-Rodella et al., 1982; Pinheiro and Schlindwein, 1998; Laporta and Suyama, 2002).

MATERIALS AND METHODS

The source of the populations studied for each analysis is given in Table 1. In each population three to five individuals were sampled.

Table 1.

Species and study sites for Ipomoea populations from Argentina, Prov. Córdoba

Species
 Population
 Flower colour
 Localities and dates of study
 Voucher
 Data taken
I. alba L. 1 White Dept Capital, Barrio Cofico, January 13, 1996. Bernardello & Galetto 890 Nectar, pollinators
2 White Dept. Capital, Barrio Cerro de las Rosas, December 12, 1987. Galetto & Bernardello 10 Nectar chemistry, nectary, pollinators
3 White Dept. Capital, Argüello, February 22, 1989. Galetto & Bernardello 49 Nectar chemistry, pollinators
I. cairica (L.) Sweet 1 Violet-pink Dept. Colón, Villa Allende, November 11, 1987. Galetto & Bernardello 8 Nectar chemistry, nectary, pollinators
2 Violet-pink Dept. Colón, El Diquecito, November 21, 1987. Galetto & Bernardello 3 Nectar chemistry, pollinators
3 Violet-pink Dept. Colón, La Quebrada, October 26, 1989. Galetto w.n. Nectar chemistry, pollinators
4 Violet-pink Dept. Colón, Río Carnero, February 13, 1993. Galetto w.n. Nectar, pollinators
5 Violet-pink Dept. Colón, Río Ceballos, 20 Dec. 1991. Galetto w.n. Nectar chemistry
I. hieronymi (O.K.) O'Donell var. hieronymi 1 Pink Dept. Capital, Villa Warcalde, 30 Dec. 1988. Galetto 38 Nectar chemistry, nectary, pollinators
2 Pink Dept. Santa María, Los Aromos, 29 Dec. 1996 Galetto 717 Nectar, pollinators
I. indica (Burm. f.) Merr. 1 Blue Dept San Justo, Miramar, 24 May 1995 Galetto & Bernardello 322 Nectar secretion, pollinators
2 Blue Dept Capital, Villa Warcalde, 30 Dec. 1988 Galetto & Bernardello 40 Nectar chemistry, nectary, pollinators
3 Blue Dept Capital, Barrio Escobar, 19 Dec. 1987 Galetto & Bernardello 11 Nectar chemistry, pollinators
4 Blue Dept Capital, Cerro de Las Rosas, 21 Dec. 1988 Galetto & Bernardello 35 Nectar chemistry
I. purpurea (L.) Roth 1 Purple Dept Santa María, La Serranita, 7 Feb. 1997 Galetto 671 Nectar, pollinators
2 Pink Dept Capital, Córdoba, 8 June 1997 Galetto 725 Nectar, pollinators
3 Purple Dept Capital, Córdoba, 8 June 1997 Galetto 726 Nectar, pollinators
4 White Dept Capital, Barrio Escobar, 5 Apr. 1988 Galetto & Bernardello 12 Nectar chemistry, nectary
5 Purple Dept Capital, Barrio Escobar, 7 Apr. 1988 Galetto & Bernardello 13 Nectar chemistry, pollinators
I. rubriflora O'Donell 1 Red Dept Punilla, Carlos Paz, 22 Mar. 1989 Galetto 52 Nectar chemistry, nectary
2 Red Dept Santa María, La Serranita, 16 Mar. 1997 Galetto 714 Nectar secretion and chemistry
3 Red Dept Colón, Río Carnero,13 Feb. 1993 Galetto w.n. Nectar secretion and chemistry
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Vouchers are deposited at CORD (Museo Botanico de Córdoba).

w.n., Without number.

In each population studied, flower visitors were recorded on individual plants at the middle of the lifetime of the flower, for 15 min on three different days.

To analyse nectary structure, flowers were fixed in 70 % ethanol, dehydrated in an ethyl alcohol–xylol series, and embedded in Paraplast. Cross- and longitudinal sections were cut at 10 µm, mounted serially, stained with safranin– astral blue (Maacz and Vagas, 1961), and observed with a compound microscope at ×100–1000 magnifications. To detect stomata in the nectariferous tissue, glands were cleared with standard bleach for 1 min and stained with Lugol solution (I2/IK). Photomicrographs were taken with Kodak T-Max film, 100 ASA, with an Axiophot-photomicrographic system equipped with automatic exposure.

Flower length, excluding the pedicel, was measured (n = 10 flowers per species). Nectary tissue volume was calculated with the non-circular section toroids' formula: V = 2πsr, where s = nectary sectional area and r = nectary radius measured from the sections' centre of gravity (n = 4 flowers per species). This parameter was estimated with reference to the weight of the drawings of the nectary in longitudinal section (the two stained areas at the ovary base). The drawings were made on a homogeneous paper using a camera lucida fitted on a stereomicroscope.

Ovaries for observation under a scanning electron microscope (SEM) were dehydrated in an acetone series and dried using CO2 in a critical-point dryer (Balzers, Switzerland). Dried samples were mounted and then gold-coated to a thickness of 25 nm (Balzers). Photomicrographs were taken with a Jeol 35CF scanning electron Photomicroscope on AgfaPan APX 100.

In the field, nectar drops for each sample were placed in 1 mL vials and quickly frozen. Sugar concentration and nectar volume were measured in the field with an Atago pocket refractometer and graduated capillary glass tubes, respectively. Tests following Baker and Baker (1975) for amino acids, lipids, phenols, alkaloids and reducing acids were made on nectar spots on chromatography paper. The ‘histidine scale’ (Baker and Baker, 1975) was used to quantify amino acids. Sugars were identified via gas chromatography. Nectar was lyophilized and silylated according to Sweeley et al. (1963). The derivatives were then injected into a Konik KNK 3000-HRGS gas chromatograph equipped with a Spectra-Physics SP 4290 data integrator, a flame ionization detector and an OV 101 column (2 m long and 3 mm diameter, on 3 % Chromosorb G/AW-DMCS mesh 100–120). Nitrogen was the carrier gas and the following temperature programme was used: 208 °C/2 min, 1 °C/min until 215 °C, 8 °C/min until 280 °C for 5 min. Carbohydrate standards (Sigma Chemical, St Louis, MO, USA) were prepared using the same method. Chromatographic sugar analyses were run at least twice for each sample. Sugar ratios, r = sucrose [S] : (fructose [F] + glucose [G]), and hexose ratios, h = G : F, were calculated as per Baker and Baker (1983b).

Floral longevity was determined in ten bagged flowers by following the flowers' development until the corollas began to wilt. Randomly chosen flowers in the bud stage were bagged using paper bags to prevent pollinator visits and were tagged for identification. Nectar production was determined by using flower sets of seven to 20 flowers each according to flower availability. Flowers of a set were assigned from different individuals (one to four per each plant according to the availability of plants). The sampling schedule took into account the lifetime of the flower of each species, with either four or five flower sets (Table 4). Data were taken once for each set, allowing the nectar to accumulate until it was measured. Net nectar production rate (NPR) per hour was calculated as: mg of sugar produced between measurements/number of hours between them (mg h−1).

Table 4.

Nectar sugar concentration (% of sucrose, wt/wt), nectar volume (µL), and mg of sugar of six Argentinian Ipomoea species measured in flower sets subjected to different removal schedules throughout the lifetime of the flower

(a) I. alba (each flower set n = 10)
Time of sampling (hours after flower opening)
2000 (1)
 2300 (4)
 0200 (7)
 0500 (10)
 0800 (13)
 Total amount of sugar per flower (mg)
Set 1
    Conc. 35·14 ± 1·63 6·42 ± 2·76 19·00 ± 3·26 14·42 ± 1·81 13·50 ± 3·90 17·31 ± 5·65
    Volume 10·28 ± 1·57 31·14 ± 16·0 9·85 ± 3·23 11·85 ± 13·7 13·50 ± 3·90
    mg sugar 3·98 ± 0·58 9·22 ± 4·74 1·98 ± 0·67 1·81 ± 2·22 0·32 ± 0·29
Set 2
    Conc. 36·14 ± 0·75 28·07 ± 1·30 21·21 ± 1·82 17·14 ± 2·26 21·06 ± 4·94
    Volume 31·42 ± 0·69 17·42 ± 9·10 4·64 ± 1·18 8·71 ± 10·87
    mg sugar 13·06 ± 5·30 5·49 ± 2·79 1·09 ± 0·35 1·41 ± 1·86
Set 3
    Conc. 34·42 ± 1·94 27·85 ± 2·54 23·07 ± 2·49 22·15 ± 7·80
    Volume 46·28 ± 1·90 11·42 ± 13·9 3·14 ± 0·89
    mg sugar 17·88 ± 5·07 3·45 ± 3·96 0·81 ± 0·24
Set 4
    Conc. 34·14 ± 1·87 25·35 ± 2·71 17·69 ± 3·76
    Volume 43·71 ± 1·75 3·00 ± 0·57
    mg sugar 16·84 ± 3·87 0·84 ± 0·19
Set 5 (control)
    Conc. 31·06 ± 2·02 19·41 ± 4·30
    Volume 50·12 ± 2·02
    mg sugar 19·41 ± 4·30
(b) I. cairica (each flower set n = 7)
Time of sampling (hours after flower opening)
0930 (2)
 1230 (5)
 1430 (7)
 1700 (9·5)
 Total amount of sugar per flower (mg)
Set 1
    Conc. 52·10 ± 5·64 46·70 ± 3·77 28·44 ± 2·60 29·33 ± 4·45 5·05 ± 0·49a
    Volume 1·55 ± 0·43 4·70 ± 0·73 3·25 ± 1·71 0·90 ± 0·99
    mg sugar 1·00 ± 0·32 2·63 ± 0·32 1·14 ± 0·39 0·29 ± 0·29
Set 2
    Conc. 52·60 ± 5·56 35·00 ± 5·92 30·87 ± 4·01 5·46 ± 0·75a
    Volume 4·20 ± 0·78 5·40 ± 1·50 1·75 ± 1·11
    mg sugar 2·72 ± 0·46 2·14 ± 0·29 0·61 ± 0·39
Set 3
    Conc. 47·60 ± 5·75 36·80 ± 3·85 3·84 ± 0·65b
    Volume 5·10 ± 1·97 2·30 ± 0·92
    mg sugar 2·89 ± 1·04 0·96 ± 0·35
Set 4 (control)
    Conc. 44·70 ± 5·39 4·18 ± 1·09b
    Volume 8·00 ± 5·39
    mg sugar 4·18 ± 1·09
(c) I. hieronymi var. hieronymi (each flower set n = 7)
Time of sampling (hours after flower opening)
0800 (1)
 1030 (3·5)
 1300 (6)
 1530 (8·5)
 Total amount of sugar per flower (mg)
Set 1
    Conc. 34·42 ± 4·45 26·31 ± 6·15 29·21 ± 4·22 22·44 ± 3·59 3·13 ± 0·25a
    Volume 1·35 ± 0·41 1·75 ± 0·74 2·81 ± 1·37 5·20 ± 1·52
    mg sugar 0·55 ± 0·24 0·50 ± 0·43 0·89 ± 0·28 1·19 ± 0·37
Set 2
    Conc. 38·14 ± 1·76 31·00 ± 4·42 28·72 ± 6·66 3·00 ± 0·41a
    Volume 2·28 ± 0·67 2·14 ± 1·09 4·07 ± 0·92
    mg sugar 1·01 ± 0·30 0·69 ± 0·22 1·30 ± 0·45
Set 3
    Conc. 37·66 ± 3·07 29·55 ± 4·82 2·53 ± 0·61a
    Volume 2·70 ± 0·75 4·00 ± 1·61
    mg sugar 1·19 ± 0·37 1·33 ± 0·40
Set 4 (control)
    Conc. 36·64 ± 4·04 5·01 ± 2·11b
    Volume 12·00 ± 5·21
    mg sugar 5·01 ± 2·11
(d) I. indica (each flower set n = 10)
Time of sampling (hours after flower opening)
0800 (1)
 1100 (4)
 1400 (7)
 1700 (10)
 2000 (13)
 Total amount of sugar per flower (mg)
Set 1
    Conc. 34·83 ± 2·22 34·83 ± 2·22 36·33 ± 1·86 31·5 ± 2·51 23·25 ± 4·03 5·47 ± 1·19a
    Volume 3·00 ± 0·63 4·33 ± 0·2 3·33 ± 1·03 2·41 ± 2·06 0·75 ± 0·75
    mg sugar 1·21 ± 0·30 1·73 ± 0·34 1·44 ± 0·47 0·89 ± 0·79 0·20 ± 0·23
Set 2
    Conc. 38·50 ± 1·51 8·16 ± 2·99 2·33 ± 2·42 8·50 ± 1·22 6·07 ± 0·74a
    Volume 5·66 ± 1·51 3·33 ± 0·81 3·75 ± 0·75 2·00 ± 1·09
    mg sugar 2·55 ± 0·35 1·48 ± 0·35 1·40 ± 0·40 0·64 ± 0·37
Set 3
    Conc. 39·83 ± 2·04 35·16 ± 2·13 3·66 ± 4·80 5·75 ± 0·79a
    Volume 6·66 ± 0·81 3·66 ± 0·51 4·33 ± 3·14
    mg sugar 3·11 ± 0·44 1·50 ± 0·22 1·13 ± 0·79
Set 4
    Conc. 40·00 ± 1·41 33·50 ± 2·81 4·77 ± 0·85ab
    Volume 8·00 ± 1·55 2·58 ± 0·49
    mg sugar 3·76 ± 0·70 1·01 ± 0·22
Set 5 (control)
    Conc. 36·83 ± 1·72 3·98 ± 0·69b
    Volume 9·33 ± 1·63
    mg sugar 3·98 ± 0·69
(e) I. purpurea (each flower set n = 15)
Time of sampling (hours after flower opening)
0830 (1)
 1130 (4)
 1430 (7)
 1730 (10)
 Total amount of sugar per flower (mg)
Set 1
    Conc. 38·00 ± 4·17 25·30 ± 5·50 – – 1·50 ± 0·23a
    Volume 2·40 ± 0·45 1·30 ± 0·80 0 0
    mg sugar 1·05 ± 0·15 0·45 ± 0·35 0 0
Set 2
    Conc. 40·00 ± 1·70 23·20 ± 1·6 – 1·55 ± 0·62a
    Volume 3·10 ± 0·76 0·46 ± 0·32 0
    mg sugar 1·35 ± 0·51 0·20 ± 0·21 0
Set 3
    Conc. 41·50 ± 6·37 – 1·32 ± 0·28a
    Volume 2·85 ± 0·83 0
    mg sugar 1·32 ± 0·28 0
Set 4 (control)
    Conc. 38·20 ± 7·59 1·24 ± 0·12b
    Volume 2·42 ± 0·82
    mg sugar 1·24 ± 0·12
(f) I. rubriflora (each flower set n = 20)
Time of sampling (hours after flower opening)
0830 (1·5)
 1130 (4·5)
 1330 (6·5)
 1530 (8·5)
 Total amount of sugar per flower (mg)
Set 1
    Conc. 25·15 ± 2·20 19·72 ± 5·51 – – 0·80 ± 0·39
    Volume 2·43 ± 0·88 0·64 ± 0·02 0 0
    mg sugar 0·67 ± 0·27 0·13 ± 0·16 0 0
Set 2
    Conc. 24·98 ± 2·42 – – 0·90 ± 0·33
    Volume 3·16 ± 0·99 0 0
    mg sugar 0·90 ± 0·33 0 0
Set 3
    Conc. 25·17 ± 2·50 – 0·88 ± 0·33
    Volume 3·11 ± 1·12 0
    mg sugar 0·88 ± 0·33 0
Set 4 (control)
    Conc. 25·42 ± 2·17 1·03 ± 0·49
    Volume 3·73 ± 1·73
    mg sugar 1·03 ± 0·49
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Data on the diagonal (first measurements of each set underlined) correspond to the nectar production dynamics of each species.

The total amount of sugar per flower was calculated for each set using set 4 or 5 as control, according to the species. When statistically significant differences were found, lower-case letters as superscript indicate a posteriori test results.

To evaluate the effect of removal on total sugar amount, nectar was removed and measured from the same flower repeatedly during the entire active secretion period. Nectar was extracted with capillary glass tubes without removing the flowers from the plant, taking extreme care to avoid damage to the nectaries. Sets of seven to 20 flowers were subjected to a different number of removals according to the secretion period of the species. Flowers of a set were assigned from different individuals (one to four per each plant according to the availability of plants). According to the flower lifetime of the species, four or five flower sets were assigned (see Table 4); for the first measurement, nectar was allowed to gather for approx. 1 h because it was not secreted in buds, and an interval of approx. 3 h was left to allow nectar to accumulate between measurements. The general scheme was to allow nectar to accumulate for a determined period (approx. 3, 6, 9, 12 h, according to the set; see Table 4) and then to remove it a number of times: set 1 = five to four nectar removals; set 2 = four to three removals; set 3 = three to two removals; set 4 of the species that have five sets = two removals; set 4 and set 5 (control sets, Table 4) = nectar was allowed to accumulate during the entire flower lifetime and only one measurement was performed. The total amount of sugar per flower was calculated as the product of nectar volume and sugar concentration per unit volume, e.g. mg per µL after Bolten et al. (1979).

Statistical tests were performed using methods described in Sokal and Rohlf (1995) with the SPSS statistical program package (SPSS release 10.0, 1999). All distributions were tested for randomness of nominal data (Runs test), homogeneity of variances (Levene test), and departures from normality (Kolmogorov–Smirnov for goodness-of-fit test). The effects of nectar removal on the total amount of sugar produced by each set of flowers were compared with one-way analysis of variance (ANOVA) and with the Bonferroni's post hoc test for multiple comparisons among pairs of means, to evaluate the consequences of pollinator visits to each species. Regression analyses were done using species means to estimate if flower traits are explained by the increase of flower size. The relationship between pollination guilds and nectar traits was assessed by qualitative comparisons because of the low number of hummingbird and hawk-moth species.

RESULTS

Floral visitors

Hymenopterans were regular visitors of I. cairica, I. hieronymi, I. indica and I. purpurea (Table 2). The introduced European bee (Apis mellifera) was occasionally observed on the study species, but most visits corresponded to native bees from the families Apidae (Bombus opifex, B. morio), Megachilidae (Megachile sp.), Anthophoridae (Centris sp., Thygater sp.) and Halictidae (Anglochloropsis sp.) (Table 2). In the populations of I. cairica and I. purpurea studied, both Bombus species were more frequent visitors than the other bees. On the other hand, hummingbirds [Trochilidae, both sexes of Chlorostilbon aureoventris and females of Sappho sparganura] were the most frequent visitors of I. rubriflora and sphingids (Sphingidae: Manduca sp. and Agrius cingulata] of I. alba (Table 2).

Table 2.

Flower visitors of Argentinian Ipomoea species

Species
 Hymenopterans
 Hummingbirds
 Sphingids
I. alba – – Sphingidae: Manduca sp. ***, Agrius cingulata***
I. cairica Apidae: Bombus opifex***, B. morio***, Apis mellifera* – –
Megachilidae: Megachile sp.**
Anthophoridae: Centris sp.**, Thygater sp.**
Halictidae: Anglochloropsis sp.**
I. hieronymi Apidae: Bombus opifex*, B. morio* – –
Megachilidae: Megachile sp.**
Anthophoridae: Centris sp.*, Melitoma sp.***, Thygater sp.***
Halictidae: Anglochloropsis sp.**
I. indica Apidae: Bombus opifex*, B. morio***, Apis mellifera* – –
Anthophoridae: Thygater sp.***
Halictidae: Halictus sp.**
I. purpurea Apidae: Bombus opifex***, B. morio***, B. bellicosus *, Apis mellifera* – –
Megachilidae: Megachile sp.**
Anthophoridae: Thygater sp.**, Ptilothrix sp.*
Halictidae: Halictus sp.*
I. rubriflora Apidae: Bombus opifex* Trochilidae: Chlorostilbon –
Vespidae: Polystes canadensis* aureoventiris***, Sappho sparganura***
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Frequency: *, rare, **, common, ***, very common, –, not recorded.

Floral nectaries

All the species studied had a conspicuous floral (also called nuptial), discoidal nectary surrounding the ovary base (Figs 1A and C; 2A, B, D, E and G). The secretory tissue was composed of intensely stained cells, each with a big nucleus and many small vacuoles (Fig. 2C, F and I), and was supplied by vascular bundles with both xylem and phloem branches (Fig. 2A and H–I). The epithelial cells possessed a cuticle, fewer cellular contents, and generally a big vacuole (Fig. 2I). Numerous, always open, stomata were found on the epidermis of the nectaries (Fig. 1B and D); nectar exudation possibly occurs through them. Their distribution varied according to the species: homogeneously distributed over all the nectary surface (I. indica and I. rubriflora, Fig. 1A and B), in two areas of the nectary: the apex and in the base (I. alba), or exclusively in the apical region (I. cairica, I. hieronymi and I. purpurea; Fig. 1C and D).

Fig. 1.

Fig. 1.

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Nectary SEM photomicrographs: (A and B) Ipomoea rubriflora, (C and D) I. purpurea. (A and C) View of ovary with the nectary surrounding its base; arrow head points to one stoma. (B and D) Detail of the nectary epidermis showing several stomata. Scale bars: A and C = 500 µm; B and D = 50 µm.

Fig. 2.

Fig. 2.

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Optical microscope photomicrographs showing nectary structure: (A–C) Ipomoea hieronymi; (D–F) I. indica; (G–I) I. rubriflora. (A) Flower partial longitudinal section; (B) flower cross-section; (C) detail of the nectariferous tissue indicated in B; (D–E) flower cross-sections at lower and upper levels of the nectary; (F) detail of the nectariferous tissue outlined in E; (G) flower partial longitudinal section; (H) detail of nectary outlined in G; (I) detail of the nectariferous tissue indicated in H. Abbreviations: k, calyx, c, corolla, n, nectary, o, ovary. Arrow heads indicate vascular bundles. A and B at the same scale; D and E at the same scale.

Floral longevity

Flowers lasted less than half a day: from 8–9 h in I. hieronymi, approx. 10 h in I. cairica, I. purpurea and I. rubriflora to approx. 12 h in I. alba and I. indica. With the exception of I. alba, whose flowers opened at twilight and faded at sunset or exceptionally at midday, the remaining species had diurnal anthesis, lasting from early morning to the afternoon.

Nectar chemical composition

Alkaloids, phenols, antioxidants and lipids were never detected. On the other hand, all samples had amino acids and sugars in variable concentrations (Table 3). Amino acids were found in concentrations from 2 to 7 on the histidine scale (Table 3). The three most common sugars were always detected and the proportions found for the different samples of each taxon were, in general, homogeneous with the exception of I. cairica that showed a great intraspecific variability (Table 3). Most species had sucrose-dominant nectars; only I. cairica presented hexose dominant or hexose-rich samples (Table 3). Hexose ratios indicated that most species had more glucose than fructose (Table 3).

Table 3.

Chemical composition of nectar in Ipomoea species

Sugars (%)
Species
 Population no.
 Sucrose
 Fructose
 Glucose
 r
 hr
 hs
I. alba 1 87·19 ± 4·84 8·78 ± 3·21 4·01 ± 2·53 6·81 0·45 5
2 63·14 ± 2·01 20·43 ± 1·84 16·42 ± 2·41 1·71 0·80 5
3 82·21 ± 3·88 13·80 ± 4·24 3·98 ± 0·97 4·62 0·28 4
Mean 77·51 ± 12·7 14·33 ± 5·84 8·13 ± 7·17 3·45 0·56
I. cairica 2 0·52 ± 0·37 34·54 ± 2·06 64·93 ± 2·42 0·005 1·87 5
5 35·62 ± 3·34 18·81 ± 1·72 45·52 ± 2·09 0·55 2·41 6
3 0·37 ± 0·30 36·52 ± 3·69 63·10 ± 3·99 0·003 1·72 2
1 0·34 ± 0·23 32·33 ± 1·68 67·32 ± 2·87 0·003 2·08 5
4 15·43 ± 2·12 36·86 ± 1·38 47·68 ± 0·75 0·18 1·29 4
Mean 10·45 ± 15·5 31·81 ± 7·49 57·71 ± 10·3 0·12 1·81
I. indica 4 55·74 ± 4·05 18·41 ± 3·92 25·84 ± 2·13 1·25 1·40 6
3 50·74 ± 6·71 21·12 ± 1·81 28·12 ± 4·90 1·03 1·33 7
2 54·31 ± 1·89 18·81 ± 2·13 26·87 ± 1·37 1·18 1·42 7
Mean 53·60 ± 2·58 19·45 ± 1·46 26·94 ± 1·14 1·15 1·39
I. hieronymi 2 60·39 ± 10·97 15·58 ± 6·22 24·01 ± 5·25 1·52 1·54 2
1 65·04 ± 5·76 11·03 ± 2·49 23·91 ± 3·27 1·86 2·17 2
Mean 62·72 ± 3·28 13·31 ± 3·21 23·96 ± 0·07 1·68 1·80
I. rubriflora 2 66·08 ± 4·98 22·13 ± 5·00 11·78 ± 2·52 1·94 0·53 6
1 64·86 ± 4·57 19·17 ± 3·88 15·96 ± 2·61 1·84 0·83 6
3 62·61 ± 8·93 13·95 ± 8·74 23·42 ± 4·33 1·67 1·67 5
Mean 64·52 ± 1·76 18·42 ± 4·14 17·05 ± 5·89 1·82 0·93
I. purpurea 4 68·71 ± 5·07 7·48 ± 2·55 23·79 ± 2·50 2·19 3·18 3
2 65·74 ± 7·52 14·01 ± 1·67 20·24 ± 3·84 1·91 1·44 3
3 69·63 ± 6·62 8·96 ± 4·00 21·41 ± 2·93 2·29 2·38 5
1 78·68 ± 2·00 10·27 ± 0·85 11·04 ± 1·14 3·69 1·07 5
5 60·08 ± 5·51 15·88 ± 3·37 24·03 ± 4·07 1·50 1·51 4
Mean 68·57 ± 6·77 11·32 ± 3·52 20·10 ± 5·31 2·18 1·78
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Values are means ± s.d.

r, Sugar ratio; hr, hexose ratio; hs, histidine scale.

Population nos corresponds to those in Table 1 and the number of individuals sampled for each one was four.

Nectar production dynamics

Nectar traits varied among the different species examined, but was quite constant for each one. Most species had a mean nectar sugar concentration thoughout the flower lifetime ranging from 34·28 to 39·42 % (I. alba: x̅ = 34·28 % ± 1·68, I. hieronymi: x̅ = 36·71 % ± 1·65, I. indica: x̅ = 37·99 % ± 2·18, I. purpurea: x̅ = 39·42 % ± 1·42). Extreme values were recorded for I. cairica with the most concentrated nectar (x̅ = 49·25 % ± 3·27), whereas I. rubriflora had the most dilute (x̅ = 25·18 % ± 0·16). Total nectar volume of unvisited flowers ranged from 50·12 µL in I. alba to 2·42 µL in I. purpurea.

In none of the species did the buds secrete nectar. Nectar secretion began as soon as the flowers opened and lasted for a few hours, as in I. purpurea and I. rubriflora, or was continuous during the whole flower lifetime in the remaining species (Table 4, underlined data on the diagonal). Ipomoea hieronymi stood out because there was a notable increase of sugar after midday (Table 4). It should be noted that nectar resorption was never detected for the species studied and that I. alba had a cessation period in the second half of the flower lifetime (Table 4). Some differences became evident when comparing the nectar production rate (NPR) between the Ipomoea species: the hawk-moth-pollinated I. alba showed the highest rate (approx. 2·5 mg h−1) during the active secretion period, whereas the hummingbird-pollinated I. rubriflora the lowest rate (approx. 0·15 mg h−1). All the bee-pollinated species (I. cairica, I. hieronymi, I. indica and I. purpurea) had a similar NPR (approx. 0·4 mg h−1).

Nectar removal effects

Independently of the effect of removing nectar on total sugar production, I. purpurea and I. rubriflora ceased to secrete nectar after a few hours, whereas the remaining species continued until the end of the flower lifetime (Table 4). After nectar removal, species showed different responses in terms of total nectar sugar produced (Table 4). In I. hieronymi, the total amount of nectar produced by flower sets subjected to removals was lower than control sets (F3,44 = 12·53, P = 0·001; Table 4), i.e. there was an inhibition of nectar sugar production. On the other hand, there was an increase in nectar sugar production in I. cairica, I. indica and I. purpurea after removals (F3,39 = 13·52, P = 0·001; F4,29 = 5·57, P = 0·002 and F3,79 = 2·77,P = 0·05, F3,39 = 4·32, P = 0·01, respectively; Table 4), whereas in I. alba and I. rubriflora removals had no effect on total nectar sugar production (F4,35 = 1·04, P = 0·40, and F3,79 = 1·42, P = 0·28, respectively; Table 4).

Regressions between flower traits and total nectar volume

Ipomoea alba with longer flower tubes correspondingly had the highest mean total nectar volume per flower and nectary volume (Fig. 3). In the remaining taxa, total volume ranged from x̅ = 2·7 µL in I. purpurea to x̅ = 12·0 µL in I. hieronymi, whereas nectary size ranged from x̅ = 0·6 mm3 ± 0·3 in I. rubriflora to x̅ = 6·9 mm3 ± 1·1 in I. hieronymi (Fig. 3).

Fig. 3.

Fig. 3.

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Quadratic regressions between flower length and nectar volume and nectary size in six Argentinian species of Ipomoea. Plotted values represent the arithmetic mean of each species (abbreviated species' names are shown).

Significant positive regressions were found indicating an increase among three parameters: the longer the flower, the more voluminous the nectary and the higher the nectar volume secreted (R2 = 0·92, P = 0·02, R2 = 0·99, P < 0·0001, respectively; Fig. 3). The number of stomata was not significantly correlated with nectary size and nectar volume (R2 = 0·25, P = 0·65; R2 = 0·45, P = 0·44, respectively).

DISCUSSION

The morphology and location of the nectaries found in the Ipomoea species studied follow the general pattern known for other representatives of the genus (e.g. Fahn, 1979; Stucky and Beckmann, 1982; Pinheiro and Schlindwein, 1998) and seems to be a conservative character for the family (Cronquist, 1981).

Studing nectar may help to determine taxonomic affinities of the species concerned and on the adaptation to the pollinators that visit the taxa. The six Ipomoea species analysed included a wide range of floral colours and visitors, a common feature for the whole genus (e.g. van der Pijl, 1954; Vogel, 1954; Schlising, 1970; Sobreira-Machado and Sazima, 1987; McDonald, 1991). Accordingly, differences in nectar features are to be expected, as found here. However, the differences found could not be related to the pollinator guild of the plants; only the hawk-moth-pollinated I. alba is typical for having higher nectar volume, as previously reported (e.g. Cruden et al., 1983; Opler, 1983). Thus, generalizations for nectar traits and pollinator relationships are precluded in these Ipomoea species.

Nectar secretion can be evaluated with regard to volume or milligrams of sugar, or both. Some authors have studied the effect of nectar removal only considering volume data and found that plants modify secretion as a function of the removals (e.g. Zimmerman and Pyke, 1986). Volume data are not enough to characterize flower costs of nectar secretion (nectar sugar production is more costly to the plant compared with water). Thus, if the sugar production is not known it is impossible to evaluate both the costs of secretion and the energetic reward value for the pollinators.

Concerning the nectar sugar composition, only I. cairica had hexose-predominant nectar, a composition preferred by short-tongued bees (Baker and Baker, 1983a). Regardless of the pollinator, almost all species had a sucrose-dominant nectar. This is an unusual result for bee-pollinated species, which commonly have hexose predominant nectars (Baker and Baker, 1983a; Galetto and Bernardello, 2003). Hummingbird-pollinated flowers tend to have sucrose-dominant nectar (e.g. Baker and Baker, 1983a; Freeman et al., 1985; Stiles and Freeman, 1993), which is confirmed for I. rubriflora among the climbers studied. Thus, no generalization regarding sugar composition and pollinator preference can be shown.

Several authors have found that taxonomically related plants showed a similar trend in their nectar sugar composition because they share common ancestors, rather than because they share the same floral visitors (e.g. Elisens and Freeman, 1988; van Wyk, 1993; Galetto et al. 1998; Perret et al., 2001; Torres and Galetto, 2002). In Ipomoea, two extreme trends related to nectar sugar composition were observed, hexose or sucrose predominant, but they cannot be related to the pollinators or to phylogenetic constraints; however, considering the high number of species in the genus and the scarcity of taxa studied, more data are needed to understand the significance of these results.

Differences were also found among the Ipomoea species studied here in terms of nectar production dynamics. Most of them secreted nectar continuously during the whole lifetime of the flower, whereas I. purpurea and I. rubriflora (the species that has the smallest quantity of nectar per flower) secreted most of it during the first hour of the flower lifetime. Previous data on a few other Ipomoea species, although scant, agree on the whole with the findings reported here (Real, 1981; Stucky and Beckmann, 1982; Devall and Thien, 1989). In particular, I. batatas (Real, 1981) showed similar production dynamics to I. hieronymi studied here. In contrast, in I. pandurata (Stucky and Beckmann, 1982) nectar began to be secreted in the bud and had a comparatively large total volume for a bee-pollinated plant (cf. Opler, 1983). Nevertheless, nectar production dynamics and removal effects, together with sugar composition, could not be clearly related either to the pollinator guild or the breeding system of the species involved. The SI species are all bee-pollinated (I. hieronymi, I. indica, I. cairica) and showed a similar total nectar production, but had differences in their nectar composition (I. hieronymi and I. indica are sucrose-dominant, whereas I. cairica is hexose-dominant). The SC species (I. alba, I. purpurea, I. rubriflora) showed no variation in nectar composition (sucrose-dominant nectars), but significant differences in their nectar production pattern.

In contrast, flower length was associated with both nectary size and total amount of nectar produced. Recent studies suggest that flower morphology is evolutionarily more labile and that corolla traits can frequently change (e.g. Cubas et al., 1999; Harrison et al., 1999) in comparison to changes in the nectar features (e.g. van Wyk, 1993; Galetto et al., 1998; Perret et al., 2001; Torres and Galetto, 2002). The association found here between flower size and total nectar volume secreted in Ipomoea suggests that structural constraints may play a major role in conserving nectar traits, at least in volume.

Acknowledgments

We thank two anonymous reviewers for useful comments on the manuscript, CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), ‘Secretaría de Investigaciones Científicas y Técnicas de la Universidad Nacional de Córdoba’, CONICOR (Consejo Investigaciones Científicas y Técnicas de la Provincia de Córdoba), ‘Agencia Córdoba Ciencia’, and ‘Agencia Nacional de Promoción Científica y Técnica’ for funding, and Cristina Ciarlante for help with the figures. The authors are members of CONICET.

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