Asymmetrical Legitimate Pollination in Distylous Palicourea Demissa (Rubiaceae): the Role of Nectar Production and Pollinator Visitation

Asymmetrical legitimate pollination in distylous Palicourea demissa (Rubiaceae): the role of nectar production and pollinator visitation

Hamleth Valois-Cuesta∗†,1, Pascual J. Soriano† and Juan Francisco Ornelas‡

∗ Programa de Biolog´ıa con Enfasis´ en Recursos Naturales, Facultad de Ciencias Basicas,´ Universidad Tecnologica´ del Choco,´ A.A. 292 Quibdo,´ Choco,´ Colombia † Postgrado en Ecolog´ıa Tropical, Instituto de Ciencias Ambientales y Ecologicas,´ Facultad de Ciencias, Universidad de Los Andes, Merida´ (5101), Venezuela ‡ Departamento de Biolog´ıa Evolutiva, Instituto de Ecolog´ıa A.C., Carretera antigua a Coatepec No. 351, El Haya, Xalapa, Veracruz 91070, Mexico´ (Accepted 7 March 2011)

Abstract: We investigated morph differences in attributes that contribute to rewarding floral visitors of the distylous shrub Palicourea demissa at La Mucuy cloud forest in Venezuela. In both morphs, we measured nectar production from flowers subjected to repeated removals at 2-h intervals (10 plants per morph) and flowers that accumulated nectar for 24 h (10 plants per morph). In both cases, floral visitors were excluded. In addition, we quantified nectar availability (30 plants per morph), floral visitation (10–12 plants per morph) and legitimate pollination (30 plants per morph) throughout the day. We explored morph differences in the variables mentioned above using analyses of variance, and the effects of nectar variation on floral visitation and legitimate pollination using regression models. We observed 1205 floral visits, grouped into six hummingbird (94.7%) and three insect species (5.3%), across observations (264 h). Coeligena torquata was the most frequent floral visitor (34%) in both morphs (1.4–1.7 visits per plant h−1). Nectar production and availability, and visitation rate were similar between morphs. Visitation rate and legitimate pollen deposition increased with the nectar production in both morphs, but levels of legitimate pollination were higher on short-styled flowers than long-styled flowers. These results show that short-styled and long-styled flowers reward floral visitors equally, but frequency and foraging behaviour of long-billed pollinators can promote asymmetrical legitimate pollination.

Key Words: cloud forest, disassortative pollination, distyly, hummingbirds, La Mucuy Bird Observatory, nectar production, Palicourea demissa, Venezuela

INTRODUCTION transfer of pollen between the anthers and stigmas of different morphs (Lau & Bosque 2003, Lloyd & Heterostyly is defined as the occurrence in a species Webb 1992). However, this breeding system does not of two (distyly) or three (tristyly) floral morphs that function perfectly because large quantities of self- and exhibit reciprocal herkogamy, where herkogamy is the intra-morph pollen can be found on stigmas (Barrett spatial separation of pollen presentation and pollen 1992, Garc´ıa-Robledo 2008, Hernandez´ & Ornelas receipt within or between flowers of an individual plant 2007, Lloyd & Webb 1992, Ornelas et al. 2004a). (Barrett 1992, Barrett & Shore 2008, Darwin 1877). Although morph-specific differences in several features Heterostyly is often associated with other physiological (mainly related to pollen and stigma characteristics) and structural characters, such as self- and intra-morph have been described in distylous species, the functional incompatibility and pollen and stigma polymorphisms significance of these differences, if any, is mostly not (Barrett 1992, Dulberger 1992). Darwin (1877) understood (Dulberger 1992, Ganders 1979). According hypothesizedthatthestructuralfeaturespromoteefficient to Darwin’s hypothesis, pollen from different stamen pollen transfer between floral morphs (disassortative levels should adhere to various parts of the animal’s pollination). Several workers have demonstrated that body corresponding to the position where compatible the heterostylous morphology provides for the efficient stigmas would contact the animal. Yet, because cross- pollination is necessary for reproductive success in 1 Corresponding author. Email: [email protected] both morphs, they should not differ in attributes that 394 HAMLETH VALOIS-CUESTA ET AL. contribute to attracting and rewarding floral visitors, is morphologically distylous and self- and intra-morph leading to equal reproductive success. Nevertheless, in incompatible (Valois-Cuesta et al. 2011). Despite the several distylous, self-incompatible species an asymmetry distylous condition, morph differences in most ancillary in nectar production and availability between morphs floral polymorphisms and spatial reciprocity of the sexual generates the ecological conditions for differential organshavebeendocumented(Valois-Cuestaetal.2011). visitation disrupting disassortative pollination and, as SS flowers display larger corollas, stigmatic lobes and a consequence, produces morph differences in female pollen grains than LS flowers. Similarly, the anther- reproductive success (Leege & Wolfe 2002, Ornelas et al. stigma separation (intra-morph herkogamy) is greater in 2004a, 2004b). SS than LS flowers. However, the position of anthers of Palicourea demissa Standl. is a distylous shrub chiefly LS flowers and stigmas of SS flowers is highly reciprocal, pollinated by hummingbirds (Valois-Cuesta & Novoa- whereasintheoppositedirectionthereciprocityisreduced Sheppard 2006). Under controlled cross-pollination (Valois-Cuesta et al. 2011). Although SS flowers produce conditions, Valois-Cuesta et al. (2011) observed that larger pollen grains than LS flowers, floral morphs do floral morphs do not differ in fruit set. However, short- not differ in the number of pollen grains per flower and styled plants produce 20% more fruits than long-styled inflorescence (Valois-Cuesta et al. 2011). In the studied plants under natural conditions suggesting that short- population,floralmorphsarenotspatiallyaggregatedand styled plants have a higher reproductive success through showa1:1morphratio(Valois-Cuesta&Novoa-Sheppard female function. Given that P. demissa is morphologically 2006). Most flowering occurs between May and June; distylous with morph differences in most ancillary floral each plant displays 23.4 ± 5.92 inflorescences and each polymorphisms and reciprocity of the sexual organ inflorescence opens 2.4 ± 0.1 flowers d−1 (Valois-Cuesta heights, we hypothesized that morph differences in nectar et al. 2011). The life span of individual inflorescences production would translate into differential pollinator from the first open flower until fruit initiation of last open visitation and, consequently, entail a reproductive cost. flower is about 47.6 ± 3.0 d−1 (N = 120 observations; Because short-styled (SS) flowers of P. demissa are larger H. Valois-Cuesta unpubl. data). Flowers are yellow, than long-styled (LS) flowers, SS flowers are expected to pedicellate and gamopetalous, anthesis start between produce more nectar and receive more visits than LS 05h30 and 07h30 h and last c. 14 h (Valois-Cuesta & flowers. If pollinator visitation rates and legitimate pollin- Novoa-Sheppard 2006). Flowers produce nectar and are ation increase with increased nectar production and these visited by hummingbirds and insects (Valois-Cuesta & relationships differ between floral morphs, we also pre- Novoa-Sheppard 2006). dicted that disassortative pollination would be disrupted.

Natural patterns of nectar production METHODS The amount of nectar secreted by a flower may not Study site be clearly revealed when natural patterns of nectar production are measured, particularly among species Fieldwork was carried out in the cloud forest that respond positively to nectar extraction by their that surrounds La Mucuy Bird Observatory (8◦38N, pollinators (Castellanos et al. 2002, Ornelas et al. 2004b). 76◦02W; 2300–2400 m asl), in the Sierra Nevada We quantified nectar production to determine whether National Park, Merida,´ Venezuela. In the study area, the plantsofeachmorphrewardpollinatorsequallyandwhen mean annual temperature is 14 ◦C and the mean annual the pollinators sought out such resources (Ornelas et al. precipitation ranges between 2800 and 3400 mm, 2004b). Inflorescences of 20 plants (10 of each morph) with peaks in April–May and October–November. were bagged in May 2008 with tulle before bud opening. Moreover, mean annual horizontal precipitation (fog) Nectar was extracted the following day with graduated is about 300 mm (Ataroff & Rada 2000). This cloud micropipettes (20 μL) without removing the flowers from forest area characterized by tree-ferns is rich in vascular the plant (non-destructive method). Nectar production epiphytes (Ataroff & Sarmiento 2004) and bird species; was measured repeatedly throughout the life of individual about 14 hummingbird species have been recorded in the flowers at 2-h intervals (at 08h00, 10h00, 12h00, area (Rengifo et al. 2005). 14h00, 16h00 and 18h00) to minimize the effects of evaporation in the quantification of nectar production. Fifty-nine flowers were examined (29 SS and 30 LS). In Study species the same plants, we measured the nectar for which buds (40 SS and 44 LS) of selected inflorescences were excluded Palicourea demissa Standl. (Rubiaceae) is a common shrub from floral visitors to let nectar accumulate for 24 h. The (3–8 m in height) at La Mucuy. The studied population accumulated nectar was sampled once in each flower at Nectar and legitimate pollination in Palicourea (Rubiaceae) 395

18h00 on the day after the exclusion. Nectar volume was Statistical analyses measured as described. We used repeated-measures ANOVAs to analyse differences between morphs in terms of nectar production Nectar standing crop (nectar availability) (volume) and visitation rate. Between-subject variation was assessed with floral morph, plant, type of floral visitor Because pollinators are responding to nectar standing (hummingbirdvs.insectspecies),visitorspecies(allvisitor crop, we also extracted the nectar available in flowers species), hummingbird species and insect species as the that had been exposed to floral visitors and measured its main factors. The model includes the effects of time of day volume and nectar concentration. Data were collected as repeated factor (within-subject variation). The sum of from flowers of 30 randomly selected plants of each total nectar produced (volume) after repeated removal morph (362 flowers of each morph) in May 2008. was compared with that of flowers in which nectar was Nectar was collected at six different times (2-h intervals, allowed to accumulate for 24 h using a two-way ANOVA. from 06h00 to 18h00) to evaluate eventual variation To describe the effects of floral morph and time of day in sugar through the peak of hummingbird activity. on nectar standing crop (volume, sugar concentration, Nectar volume was measured as described and sugar mass of sugar) and legitimate pollen deposition, nested concentration (percentage sucrose) was determined with two-way ANOVAs were used. Floral morph and time of a pocket refractometer (Bellingham + Standley 45–81, day were treated as fixed factors. Among-plant variation Buffalo, New York, USA; range concentration 0◦–50◦ was assessed with floral morph and plant as the main BRIX scale). The amount of sugar produced was expressed factors. Along with the main effects, a floral morph × in milligrams after Bolten et al. (1979) and Kearns & time of day interaction was included in the model. Data Inouye (1993). were log(x+1)- or square root-transformed to correct normality and homoscedasticity, but untransformed data (mean ± SE) are reported. Morph differences in the number of visits per plant were assessed with a Mann– Floral visitors Whitney test, and the number of probes per visit and visitation rate per plant from each floral visitor were We conducted field observations (264 h) on 22 assessed using one-way ANOVAs. To evaluate species randomly selected plants (10 SS and 12 LS) throughout richness in the foraging guild of P. demissa, a species- the blooming peak (May–June 2008) to describe the accumulation curve was performed using the software assemblage composition and foraging modes of floral EstimateS 7.5 developed by Robert K. Colwell (available visitors and to determine daily patterns of floral visitation. at http://purl.oclc.org/estimates) with flower visitors as We watched each focal plant for 12 h, from 06h00 a function of our observational sampling effort. Lastly, to 18h00. For each plant, we recorded species, time of data on legitimate pollen deposition and visitation rate visitation, number of probes per visit, and the number of per plant were also analysed using quadratic regression flowers visited per foraging bout of each floral visitor. analyses for nectar production (total volume of secreted nectar per flower) and availability (nectar volume, sugar concentration and amount of sugar of flowers available to Legitimate pollen deposition floral visitors) from 10 LS and 10 SS plants of P. demissa. Regression models in each relation were evaluated using To assess legitimate pollination between morphs, we ANOVAs and the slopes were compared between LS and randomly collected 360 flowers (180 flowers from 30 SS plants using t-test. All statistical analyses were run plants of each morph) exposed to their pollinators at six using SPSS 12.0 (SPSS, Inc. Chicago, IL, USA). different times (2-h intervals, from 08h00 to 18h00). The stigma of each flower exposed to natural pollination was carefully removed and mounted on a microscope RESULTS slide (Kearns & Inouye 1993). Samples were observed under a Leitz Dialux 20 EB microscope. The magnitude Nectar production and availability of legitimate pollination was assessed by counting the number of pollen grains of the opposite morph on the Nectarproduction(volume)wasnotsignificantlydifferent stigma in both morphs. We assumed that pollen receipt between SS and LS flowers after repeated removals was an indication of pollinator activity, and that pollen (repeated-measures ANOVA; F1,195 = 3.95, P = 0.075; grains with a diameter >105 μm arrived from SS flowers Figure 1). Also, the among-plant differences were not < μ and those 100 m arrived from LS flowers (Valois- significant (F18,195 = 0.82, P = 0.66). Nectar production Cuesta et al. 2011). varied significantly over time (F5,195 = 67.1, P < 0.0001) 396 HAMLETH VALOIS-CUESTA ET AL.

12 35 Short-styled 10 Short-styled Long-styled a 30 Long-styled 8

25 a b 6 4 20 2 Nectar volume (µL) 15 0 24 b 10 22

Accumulated nectar (µL per flower) 800 1000 1200 1400 1600 1800 24 20 Time of day (h) 18 Figure 1. Nectar production in short-styled (SS) and long-styled (LS) 16 flowers of Palicourea demissa at La Mucuy, Merida,´ Venezuela. Flowers 14 subjected to repeated nectar removal (a) and flowers in which nectar 12 was allowed to accumulate for 24 h (b). Data indicate mean ± SE. Sugar concentration (%) 10 2.0 c but the floral morph × time-of-day interaction was not 1.5 significant (F5,195 = 1.45, P = 0.21). Overall, SS flowers accumulated more nectar after six repeated extractions 1.0 ± μ = ± (28.7 2.1 L, N 10 plants) than LS flowers (24.6 0.5 μ = 3.22 L, N 10 plants) but these differences were Mass of sugar (mg) marginally significant (one-way ANOVA; F1,39 = 4.11, 0.0 P = 0.05). The among-plant variation was not significant 600 800 1000 1200 1400 1600 1800 Time of day (h) (F18,39 = 0.69, P = 0.8). Nectar production rate (μLper flower h−1) was higher in SS flowers (1.48 ± 0.13, N = 10 ± = Figure 2. Nectar standing crop in short-styled (SS) and long-styled (LS) plants)thaninLSflowers(1.16 0.12,N 10plants)but flowers of Palicourea demissa at La Mucuy, Merida,´ Venezuela. Nectar these differences were not significant (F1,39 = 3.02, P = volume (μL) (a), sugar concentration (%) (b), amount of sugar (mg) (c). ± 0.09). Also, the plant effect (F18,39 = 0.88, P = 0.59), Data indicate mean SE. time-of-day effect (F4,156 = 1.66, P = 0.15), and the floral morph × time-of-day interaction were not significant concentration, F6,638 = 11.3, P < 0.0001; amount (F = 2.14, P = 0.07). Over 24 h of accumulation, 4,156 of sugar, F6,580 = 52.3, P < 0.0001; Figure 2). undisturbed SS flowers accumulated more nectar Among-plant variation was not statistically significant ± μ = (27.0 2.7 L, N 10 plants) than undisturbed LS (volume, F = 0.84, P = 0.79; sugar concentration, ± μ = 58,654 flowers (24.6 2.6 L, N 10 plants) but these F = 1.17,P = 0.19;amountofsugar,F = 0.81, = = 58,638 58,580 differences were not significant (F1,64 2.51, P 0.12; P = 0.84). The floral morph × time-of-day interaction among-plant variation, F = 0.68, P = 0.81). When 18,64 was significant in volume (F6,654 = 2.56, P = 0.018) and flowers that experienced repeated removals were com- amount of sugar (F6,580 = 2.67, P = 0.014), but the pared with those undisturbed flowers that accumulated interaction for sugar concentration was not significant nectar for 24 h, the total amount of nectar secreted (F6,638 = 0.58, P = 0.75). A Tukey test showed that was not significantly different between morphs (two- differences throughout the day in nectar volume and = = way ANOVA; F1,39 1.03, P 0.31; among-plant amount of sugar were significant at 06h00 (contrast = = variation, F18,39 0.54, P 0.91). The plant effect between 06h00 and 08h00–18h00, P < 0.05; contrast = = (F58,81 0. 92, P 0.62), the nectar-treatment effect 08h00–18h00, P > 0.05) but these differences in = = × (F1,81 0.72, P 0.39) and the floral morph nectar- nectar standing crop disappeared in the afternoon = = treatment interaction (F1,81 0.02, P 0.87) were not (Figure 2). significant. In flowers exposed naturally to floral visitors, nectar standing crop measures (volume, sugar concentration, Floral visitors amount of sugar) were independent of the floral morph (two-way ANOVA; volume, F1,654 = 2.32, P = 0.19; A total of 1205 floral visits by hummingbirds and insects sugar concentration, F1,638 = 3.29, P = 0.07; amount were recorded during our observations, a good sampling of sugar, F1,580 = 3.06, P = 0.08; Figure 2) but effort according to a species-accumulation curve (Table 1, these measures were not independent of the time Figure 3). We found no significant differences between of day (volume, F6,654 = 59.9, P < 0.0001; sugar morphs in the number of visits that our focal plants Nectar and legitimate pollination in Palicourea (Rubiaceae) 397

18 Observed 16 95% confidence intervals of observed Chao 1 1,20 14 ICE 12 10 8 Saturation 6 Number of species

) 4 1

− 2 0.43 1.75 0.72 0.05 0.260.06 0.73 1.67 0.57 0.41 0.05 1.77 0.16 1.57 0.10 0.62 0.026 1.80 ± ± ± ± ± ± ± ± ± 0

12 plants, 144 h) F 0 50 100 150 200 250 300 = Time (h)

Figure 3.Species-accumulationcurveforpollinatingandnon-pollinating Non-territorial. Bill size data were taken from Hilty fauna of Palicourea demissa at La Mucuy, Merida,´ Venezuela. The species =

0.05). richness estimators (Chao 1 and ICE) were obtained from abundance > data of each ﬂoral visitor (number of visits per hummingbird and insect species). Note that 200 h of observation were necessary to record the 0.43 0.64 0.67 1.67 0.15 0.31 0.21 0.11 0.40 1.24 0.26 0.15 0.01 0.19 0.02 0.17 assemblage composition of ﬂoral visitors of P. demissa. ± ± ± ± ± ± ± ± Territorial, NT 10 plants, 120 h) LS (N = = = ±

SS (N received throughout the day (SS 55.6 9.3 visits per plant, N = 10 plants, 120 h; LS = 53.9 ± 5.5 visits per plant, N = 12 plants, 144 h; Mann–Whitney test, U = 56, P = 0.82) or in the rate at which visits occurred over time ´ erida, Venezuela. T (SS = 4.6 ± 0.8 visits per plant h−1,N= 10 plants, 120 h; LS = 4.5 ± 0.5 visits per plant h−1,N= 12 plants, 144 h; 1.0 (141) 1.16 0.8 (338) 1.44 1.8 (59) 0.36 1.0 (34) 0.43 0.8 (189) 0.72 2.3 (45) 0.48 1.2 (27) 0.02 9.4 (5) 0 0.03 2.5 (27)one-way 0.04 ANOVA; F1,20 = 0.01, P = 0.93). ± ± ± ± ± ± ± ± ± The number of ﬂowers probed per visit ranged from 2

at La Mucuy, M to 94, but in most cases (86%), flower visitors probed <20 flowers per plant. Interestingly, the less frequent floral visitors (Bombus sp. and the hummingbirds Aglaicer- cus kingi and Ocreatus underwoodii) probed on average >20 flowers per plant per visit (Table 1) but morph = Palicourea demissa differences were not statistically significant (SS 17.7 ± 0.7, N = 347; LS = 18.0 ± 0.6, N = 518; one-way ANOVA; F1,862 = 0.45, P = 0.5). The visitation rate (number of visits per plant h−1) was not significantly different between floral morphs (repeated-measures ANOVA, floral-morph effect; F1,174 = 0.06, P = 0.81; Figure 4) and the among-plant variation was not significant (F20,174 = 0.4, P = 0.98). The visitation rate varied significantly over time Foraging Bill size Number of Probed flowers per visit Visitation rate (No. of visits per plant h (F11,1914 = 1.87, P = 0.04), but the floral morph × time- of-day interaction was not significant (F11,1914 = 0.81, P = 0.62). Hummingbirds were significantly more SE. Effects of morph evaluated with one-way ANOVAs. In all cases, morph differences were not significant (P

± frequent visitors than insects to plants of both morphs (hummingbirds: SS = 4.58 ± 0.78 visits per plant −1 = = ± Fraser 1840 NT 13 231 (19.2) 17.1 h ,N 10 plants,120 h; LS 4.11 0.43 visits per Latreille 1817 29 (2.4) 6.1

Lesson 1832 NT 13plant 78 (6.5) h−1,N 23.7 = 12 plants, 144 h; insects: SS = 0.06 ± Bourcier 1847 T 18 265 (22.0) 17.8 Swainson 1827 T 20 67 (5.6) 12.2 Boissonneau 1840 NT 33 413 (34.3) 18.4 Lesson 1832 T 13 87 (7.2)0.03 23.0 visits per plant h−1,N= 10 plants, 120 h;

LS = 0.40 ± 0.22 visits per plant h−1,N= 12 plants,

Floral visitors of short-styled (SS) and long-styled (LS) plants of 144 h; type of floral visitor effect, F = 20.2, P < sp. 5 (0.4)1,174 25.3 × 0.0001; floral morph type of floral visitor interaction, (2003). Data indicate mean = = Table 1. F1,174 0.93, P 0.33; Figure 4). The hummingbird SpeciesHummingbirds Adelomyia melanogenys behaviour (mm) visits (%) per plant (Number of visits) Floral morph One-way ANOVA Aglaiocercus kingi Coeligena torquata Colibri thalassinus Heliangelus spencei Ocreatus underwoodii Insects Bombus Heliconius clysonimus Lepidoptera sp. 30 (2.5) 12.8 398 HAMLETH VALOIS-CUESTA ET AL.

10 0.9 ± 0.3) were the most frequent floral visitors and Hummingbirds a had the highest visitation rates, respectively (Table 1, 8 Insects Figure 5). On the other hand, hummingbird species 6 also differed in their foraging behaviour; for example, C. torquata, A. melanogenys and O. underwoodii were 4 not territorial, whereas H. spencei and Colibri thalassinus were very territorial and aggressive. Aglaiocercus kingi 2 defended feeding territories when numerous flowers were 0 displayed on individual plants. In addition, we frequently 10 observed that after probing a few flowers (< 3–6 flowers) b H. spencei, A. kingi and A. melanogenys (all short-billed 8 Visits per plant Visits per plant hummingbirds) perched nearby and cleaned off their bills 6 on branches before they flew towards other conspecific plants. Although hummingbird species differ in the size of 4 their bills (Table 1), all probed flowers of both morphs 2 legitimately (Figure 6), and nectar robbing was not observed during our fieldwork. 0 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Time of day (h)

Figure 4. Visitation rates of hummingbirds and insects on short-styled Legitimate pollen deposition (SS) (a) and long-styled (LS) plants (b) of Palicourea demissa at La Mucuy, Merida,´ Venezuela. Data indicate mean ± SE. Plants of SS flowers received significantly more legitimate (SS = 88.0 ± 3.56, N = 30 plants; LS = 14.9 ± 0.97, N = 30 plants; nested two-way ANOVA; F1,290 = 400.9, visitation rate was independent of the floral morph P < 0.0001) and about the same number of illegitimate = ± = (F1,100 = 0.75, P = 0.38). However, the insect visitation pollen grains on stigmas (SS 60.2 5.2, N 30 plants; = ± = = = rate was significantly higher on LS plants (F1,40 = 4.7, LS 44.7 2.7, N 30 plants; F1,290 3.55, P 0.06) P = 0.036; Figure 4). In both cases, the effect of than those plants of LS flowers. However, stigmas of time of day was statistically significant (hummingbirds, SS flowers experienced proportionately higher levels = < F11,1100 = 2.16, P = 0.015; insects, F11,440 = 3.16, P < of legitimate pollination (F1,290 247, P 0.0001). 0.0001), but the hummingbird species × time-of-day Among-plant variation was not statistically significant = = (F55,1100 = 0.97, P = 0.52) and insect species × time- (F58,290 0.88, P 0.71). Although stigmas of SS flowers of-day (F22,440 = 1.44, P = 0.09) interactions were not received proportionately more legitimate pollen over time significant. than did stigmas of LS flowers, this morph difference Although the hummingbird assembly was most varied through the day (time-of-day effect, F5,290 = 0.95, frequent as floral visitor than insect assembly P < 0.45; floral morph × time-of-day interaction, (hummingbirds = 94.7%, insects = 5.3% of visits recor- F5,290 = 3.18, P = 0.008). ded; Table 1, Figure 4), hummingbird species varied The rate at which legitimate pollen grains were significantly in their visitation rates compared with deposited on stigmas was constant over time (nested two- variation in the visitation rates of insect species (repeated- way ANOVA; time-of-day effect; F5,290 = 0.32, P = 0.9), = ± measures ANOVA; visitor species effect; F8,160 = 5.3, but differed between morphs (SS 8.33 2.9 grains −1 = = ± −1 = P < 0.0001; hummingbird species effect; F5,100 = 2.7, h ,N 180; LS 1.46 0.79 grains h ,N 180; = = × P = 0.025; insect species effect; F2,40 = 1.66, P = 0.20). F1,290 4.02, P 0.046). The floral morph time-of-day However, visitation rate of individual species was interaction was not significant (F5,290 = 0.34, P = 0.88). independent of the floral morph (visitor species × floral Again, among-plant variation was not statistically = = morph; F8,160 = 0.63, P = 0.75; hummingbird species × significant (F58,290 0.18, P 1.0). floral morph; F5,100 = 0.52, P = 0.76; insect species × Finally, regression analyses showed that visitation rate floral morph; F2,40 = 0.3, P = 0.74; Table 1; Figure 5). of floral visitors and the deposition of legitimate pollen Individual plants were homogeneous for visitation rate by are explained by nectar production (secreted volume per hummingbirds(F20,100 = 0.4,P = 0.1)butheterogeneous flower per plant) in both floral morphs. However, those for visitation rate by insects (F20,40 = 2.0, P = 0.037) . relationships were stronger for SS than LS plants. For Of the six hummingbird species that visited the flowers, nectar standing crops, the volume, concentration and Coeligena torquata (34%, 1.6 ± 0.5), Heliangelus spencei amount of sugar showed no significant relationship with (22%, 1.0 ± 0.3) and Adelomyia melanogenys (19%, legitimate pollination and visitation rate (Table 2). Nectar and legitimate pollination in Palicourea (Rubiaceae) 399

5 Short-styled Long-styled ab 4

0 5 c d 4

1 Visits per plant 0 5 e f 4

0 6 7 8 9 1011121314151617 6 7 8 9 10 11 12 13 14 15 16 17 Time of day (h)

Figure 5. Visitation rates of most frequent ﬂoral visitors of Palicourea demissa at La Mucuy, Merida,´ Venezuela. Coeligena torquata (a), Heliangelus spencei (b), Adelomyia melanogenys (c), Aglaiocercus kingi (d), Ocreatus underwoodii (e), Colibri thalassinus (f). Data indicate mean ± SE.

DISCUSSION increase the probability of disassortative pollination in P. demissa since pollinators are forced to visit more plants The role of nectar on disassortative pollination throughout their foraging routes to satisfy their energetic requirements. This interpretation is partially consistent In both floral morphs, total nectar volume secreted by with results of several works showing that visitation rates flowers subjected to repeated nectar removal was not increase with increased nectar availability but pollen different from that of flowers that accumulated nectar transfer among flowers is reduced (Cresswell 1999, de until the end of day. Although the effects of different Jong & Klinkhamer 2005, Engel & Irwin 2003, Stone & nectar removal intensities on total nectar production Thomson 1994). was not evaluated here (Ordano & Ornelas 2004), our A surprising aspect of the nectar measurements results in P. demissa suggest that the repeated visits by relationships with visitation rates and legitimate floral visitors did not stimulate replenishment of removed pollination in P. demissa was the non-significant nectar (Musicante & Galetto 2008, Pleasants 1983), and relationships of nectar availability (volume, sugar that nectar evaporation was minimal. Nevertheless, the concentration and amount of sugar) with legitimate number of visits per plant showed a positive relationship pollination or visitation rate (Table 2). These results withthenectarproductioninbothfloralmorphs(Table2). suggest that the foraging behaviour of floral visitors The significance of this relationship is expected because and, consequently disassortative pollination in P. demissa, the amount of nectar produced by individual flowers in depend more on fluctuations in the amount of nectar both morphs (∼25 μL per flower) secreted at a rate of secretedratherthanchangesonitsenergeticvalueamong ∼1.3 μL per flower h−1 allows a constant nectar reward plants. Although these results need to be experimentally over time, and plants of each morph could consequently evaluated, an alternative explanation is that the achieve a constant visitation rate over time. Likewise, a observed minimal variation in sugar concentration and nectar offer of a few microlitres per flower over time could amount of sugar does not affect pollinator behaviour 400 HAMLETH VALOIS-CUESTA ET AL.

Figure 6. Frequent ﬂoral visitors of Palicourea demissa at La Mucuy cloud forest, Merida,´ Venezuela. Coeligena torquata (a), Heliangelus spencei (b), Adelomyia melanogenys (c), Aglaiocercus kingi ♀ (d), Ocreatus underwoodii ♀ (e), Colibri thalassinus (f). Scale = 1cm. Nectar and legitimate pollination in Palicourea (Rubiaceae) 401

Table 2. Non-linear regression analysis of legitimate pollen deposition (proportion of legitimate pollen grains on stigma per plant) and floral visitation rate (number of visits per plant h−1) against nectar production (secreted volume per flower per plant) and availability (volume, sugar concentration and amount of sugar per flower per plant) for short-styled (SS) and long-styled (LS) flowers of Palicourea demissa at La Mucuy, Merida,´ Venezuela. The significance of the model is indicated and differences between the slopes for LS and SS morphs are indicated in parentheses after the variable name. ∗ P < 0.05, ∗∗ P < 0.001, NS = Not significant. Quadratic regression ANOVA 2 Variable Sample: plants, flowers, hours R Model F2,7 Nectar production – Volume Visitation rate (t19 = 2.17, P = 0.04) SS 10, 29, 120 0.88 Y = 4.1 − 0.23X + 0.008X2 35.3∗∗ LS 10, 30, 120 0.78 Y = 2.34 − 0.02X + 0.003X2 17.7∗∗ Legitimate pollen deposition (t19 = 3.20, P < 0.01) SS 10, 29, 55 0.70 Y = 0.97 − 0.04X + 0.001X2 11.7∗∗ LS 10, 30, 54 0.53 Y = 0.2 + 0.001X + 0.073X2 6.10∗ Nectar availability – Volume Visitation rate (t19 =−0.37, P = 0.71) SS 10, 140, 120 0.12 Y = 7.3 − 1.9X + 0.23X2 0.51 NS LS 10, 140, 120 0.09 Y = 3.2 + 1.9X − 0.56X2 0.34 NS Legitimate pollen deposition (t19 =−0.27, P = 0.78) SS 10, 29, 55 0.10 Y = 0.65 + 0.006X − 0.004X2 0.37 NS LS 10, 30, 54 0.05 Y = 0.3 − 0.05X + 0.01X2 0.22 NS Sugar concentration Visitation rate (t19 =−0.66, P = 0.51) SS 10, 131, 120 0.38 Y = 138 − 13.4X + 0.34X2 3.83 NS LS 10, 94, 120 0.24 Y = 98.9 − 10.1X + 0.27X2 2.44 NS Legitimate pollen deposition (t19 = 0.13, P = 0.89) SS 10, 131, 55 0.24 Y = 4.59 − 0.41X + 0.01X2 2.47 NS LS 10, 94, 54 0.11 Y = 1.17 − 0.09X + 0.002X2 1.59 NS Amount of sugar Visitation rate (t19 = 0.17, P = 0.86) SS 10, 131, 120 0.01 Y = 7.1 − 8.53X + 4.2X2 0.87 NS LS 10, 94, 120 0.04 Y = 3.9 + 3.41X − 3.14X2 0.17 NS Legitimate pollen deposition (t19 =−0.95, P = 0.35) SS 10, 131, 55 0.01 Y = 0.7 − 0.2X + 0.09X2 0.63 NS LS 10, 94, 54 0.09 Y = 0.2 + 0.23X − 0.14X2 0.37 NS

patterns. Klinkhamer et al. (2001) found that pollinators frequent floral visitors of P. demissa (hummingbirds) approached groups of plants with high nectar production visited plants regardless of their morph condition. Gender rates more often than groups with low nectar production specialization based on nectar production has been rates, if the groups were segregated. However, pollinators documented in several dichogamous species (Carlson & did not discriminate within groups when plants with high Harms 2006), but little explored in heterostylous species. and low nectar production rates were placed next to each In distylous species a differential nectar production other(Wolf&Hainsworth1990).Becausefloralmorphsof between morphs could generate differential patterns of P. demissa produce a similar number of inflorescences per floral visits, pollen deposition on stigmas, and female plant, number of flowers per inflorescences, and plants reproductive output (Leege & Wolfe 2002, Manetas & are spatially intermixed regardless of morph condition Petropoulou 2002, Ornelas et al. 2004a, 2004b). Among (< 3 m of distance among plants) within the studied distylous species for which nectar production data exist, population (H. Valois-Cuesta unpubl. data), it is unlikely nectar production equals between morphs in most cases that pollinators discriminate among plants on the basis (Palicourea petiolaris and P. fendleri, Sobrevila et al. 1983; of sugar concentration, and therefore, floral morphs of Fernandusa speciosa, Castro & Oliveira 2001; Psychotria P. demissa could experience equal rates of floral visitation poeppigiana, Coelho & Barbosa 2004, Valois-Cuesta et al. and legitimate pollen deposition. 2009; P. barbiflora, Teixeira & Machado 2004a, 2004b), In P. demissa, patterns of nectar availability (nectar except for Palicourea padifolia (Ornelas et al. 2004b) volume, sugar concentration and amount of sugar) and Fagopyrum esculentum (Cawoy et al. 2008). In P. were similar between morphs (Figure 2), suggesting that demissa, similar patterns in nectar production and nectar pollinators are similarly rewarded when visiting flowers availability should promote legitimate pollination in both of either floral morph. This explains the fact that more floral morphs and, ultimately, disassortative pollination. 402 HAMLETH VALOIS-CUESTA ET AL.

Hummingbirds as mediators of disassortative pollination legitimate pollen grains on emasculated SS flowers (40%, N = 15) than emasculated LS flowers (20%, N = 15). In The effectiveness of legitimate pollination from SS to contrast, short-billed hummingbirds (A. melanogenys and LS and from LS to SS flowers depends on how flowers H. spencei) deposited 2.3 times more legitimate pollen on and pollinators are morphologically adjusted (Lloyd & LS (50%, N = 30) than emasculated SS flowers (20%, Webb 1992, Ornelas et al. 2004a, Stone & Thomson N = 30; Z. Betancourt, P. J. Soriano, H. Valois-Cuesta & 1994). Flowers of P. demissa are dimorphic (SS flowers J. F. Ornelas, unpubl. data). display larger corollas, corolla-tube entrance width, and In both morphs of P. demissa, the visitation rate show greater stigma-anther separation than those of LS and legitimate pollination were correlated with nectar flowers; Valois-Cuesta et al. 2011) and the hummingbird production (Table 2); how then could we explain the pollinators recorded here differ markedly in bill size (Hilty observed asymmetrical pollen flow and the higher female 2003; Table 2). Our study showed that hummingbirds reproductive output of SS plants previously reported play a fundamental role as pollen vectors in P. demissa, (Valois-Cuesta et al. 2011)? The reciprocal position but hummingbird species may play a different pollinator of anthers and stigmas in distylous species has been role if they vary in bill size. interpreted as a mechanism that facilitates disassortative Several workers have evaluated experimentally the pollination (Barrett 2002, Darwin 1877, Lloyd & Webb effect of the floral morphology on pollination efficiency 1992), in which a closer reciprocity among sexual organs in distylous species. For example, Stone (1995) evaluated should enhance male (legitimate pollen donation) and theefficiencyofdistylyinpromotinglegitimatepollination female (legitimate pollen receipt) reproductive success. in emasculated flowers of Psychotria suerrensis and Several authors have shown that legitimate pollen discovered that distyly is only partially effective in transfer between floral morphs is negatively associated achieving efficient pollen donation. She found that pollen with asymmetries in the position of anthers and stigmas from LS plants was transferred more efficiently to SS (Hernandez´ & Ornelas 2007, Garc´ıa-Robledo 2008, stigmas and pollen from SS plants was dispersed in Lau & Bosque 2003, Ornelas et al. 2004a, Stone & equal amounts to stigmas of both morphs. In Palicourea Thomson 1994), i.e. the low reciprocity among sexual fendleri, Lau & Bosque (2003) found that Amazilia organs diminishes pollen deposition on legitimate stigmas tobaci hummingbirds were more efficient depositing (Barrett 2002, Lloyd & Webb 1992). In P. padifolia, legitimate pollen on stigmas of SS flowers than on those Hernandez´ & Ornelas (2007) found that pollen receipt of LS flowers. They suggested that the probability of was not affected by characters typically associated with disassortative pollination in distylous species depends the efficiency of this floral polymorphism in promoting on the relative position of sexual organs between floral disassortative pollination. They showed that at high morphs (Stone 1995). Ornelas et al. (2004a) evaluated levels of stigma-anther separation there was a significant this hypothesis in P. padifolia with stuffed hummingbirds reduction in legitimate cross-pollination compared with and found that long-billed hummingbirds transferred that at low levels of separation. However, the negative significantly more pollen from LS towards SS flowers effects of herkogamy (stigma-anther separation) were than in the opposite direction. Although asymmetrical only detected for LS plants (significant interaction pollen transfer between floral morphs seemed to favour between floral morph and stigma-anther separation). SS-morph plants of P. padifolia, the observed variation in In contrast, high values of herkogamy had a positive pollenremovalandpollenreceiptwasnotexplainedbythe effect on SS plants, which received more legitimate pollen variation in hummingbird bill morphology, nor did they grains with increased spatial separation. Accordingly, the find evidence that hummingbird bill morphology solely greater anther-stigma separation (herkogamy) observed explained the differences in fruit production between in SS flowers of P. demissa (Valois-Cuesta et al. 2011) LS and SS-morph individuals (Ornelas et al. 2004b). If might favour female reproductive success in SS flowers we consider that the bill size of a hummingbird is the and male reproductive success in LS flowers; it would determining factor in the direction of pollen flow between reduce physical interference between anthers and stigma, morphs in P. demissa, we then could infer that long- minimizes spontaneous self-pollen deposition, and at the billed hummingbirds play a major role in pollen transfer same time would facilitate accessibility to pollinators between reproductive organs at low levels (from anthers in the SS morph. Therefore, the legitimate pollen flow of LS flowers towards stigmas of SS flowers), whereas between morphs would be asymmetric, with SS plants short-billed hummingbirds would be more efficient in being more effective as receptors of legitimate pollen the transfer of pollen grains among reproductive organs and LS plants as pollen donors. To fully answer this positioned at higher levels (from anthers of SS flowers question a key factor of the pollination process between towards stigmas of LS flowers). This idea is supported morphs, the visitation frequency and foraging behaviour by preliminary work on P. demissa where long-billed of pollinators, needs to be further evaluated (Engel & hummingbirds (C. torquata) deposited twice as many Irwin 2003, Feinsinger & Busby 1987, Lasso & Naranjo Nectar and legitimate pollination in Palicourea (Rubiaceae) 403

2003, Ornelas et al. 2004a). In P. demissa,along- (RLB06-M03, RLB08-SP-1) and Consejo de Desarrollo billed, non-territorial hummingbird (C. torquata) was the Cient´ıfico, Human´ıstico y Tecnologico,´ Universidad de los most frequent floral visitor in both floral morphs. If the Andes,Merida-Venezuela(C-1557–08–01EM).StaffatLa´ hypothesis of herkogamy (stigma-anther separation) is Mucuy Bird Observatory, Sierra Nevada National Park satisfied in P. demissa, it is reasonable to suggest that and Laboratorio de Ecolog´ıa Animal “A”, Universidad the observed pollen receipt pattern, in which SS flowers de los Andes, Merida,´ Venezuela provided logistical received proportionately more compatible pollen grains support. than LS flowers, could not be attributed to the higher C. torquata visitation rates but to the morphology of its bill (Z. Betancourt, P. J. Soriano, H. Valois-Cuesta & J. F. Ornelas unpubl. data) or a combination of LITERATURE CITED bill morphology and frequency of visitation. 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