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esized that ant protection of plants may also function via the repellence of floral nectar thieves. This so called “nectar- thief protection hypothesis” (van der Pijl, ...
13 (4): 431-438 (2006)

Ant protection against herbivores and nectar thieves in Passiflora coccinea flowers1 Inara R. LEAL2, Departamento de Botânica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/no - Cidade Universitária, 50670-901, Recife, PE, Brazil, e-mail: [email protected]

Erich FISCHER, Departamento de Biologia, Universidade Federal de Mato Grosso do Sul, 79070-900, Campo Grande, MS, Brazil.

Christian KOST, Abteilung für Bioorganische Chemie; Max Planck Institut für Chemische Ökologie; Hans Knoell Str. 8; 07745 Jena, Germany.

Marcelo TABARELLI, Departamento de Botânica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/ no - Cidade Universitária, 50670-901, Recife, PE, Brazil.

Rainer WIRTH, Plant Ecology and Systematics, Department of Biology, University of Kaiserslautern, P.O. Box 3049, 67653 Kaiserslautern, Germany.

Abstract: In this study we describe the ant assemblage visiting both foliar and bracteal extrafloral nectaries (EFNs) of Passiflora coccinea, investigate the protective role provided by ants against both herbivores and nectar-thief butterflies, and analyze possible fitness benefits for the plant (i.e., fruit and seed production) resulting from the presence of ants. Experiments and observations were performed in two Amazonian terra firme forest sites. Twenty-two ant species were recorded visiting the EFNs of P. coccinea. Camponotus aff. blandus was the most frequent and subordinate among the EFN-visiting ant species, accounting for 20% of the ant attacks on simulated herbivores (termites) and 50% on butterflies. Both the number of ant individuals and the number of species were positively correlated with the combined number of buds and flowers of P. coccinea. Moreover, ant protection behaviour against termites was stronger on flowers than on leaves. Finally, seed set almost doubled among fruits originating from ant-visited flowers as compared to flowers from which ants were artificially excluded. Our results suggest a protective role of ants for flowers of P. coccinea against both herbivores and nectar thieves, improving plant reproductive success. Keywords: Amazon forest, ant–plant interaction, extrafloral nectaries, flower protection, nectar thieves, plant fitness. Résumé : Dans cette étude, nous décrivons l’assemblage de fourmis visitant les nectaires extrafloraux des feuilles et des bractées de Passiflora coccinea, nous étudions le rôle protecteur des fourmis contre les herbivores et les papillons voleurs de nectar et analysons les bénéfices possibles pour la plante, résultant de la présence de fourmis, au niveau de la production de fruits et de graines. Des expériences et observations ont été réalisées dans deux sites terrestres forestiers de l’Amazone. Vingt-deux espèces de fourmis ont été observées visitant les nectaires extrafloraux de P. coccinea. Camponotus aff. blandus était l’espèce de fourmi la plus fréquente tout en étant subordonnée aux autres fourmis visitant les nectaires extrafloraux, elle produisait environ 20% des attaques contre les herbivores simulés (termites) et 50% contre les papillons. Autant le nombre d’individus que les nombre d’espèces de fourmis étaient corrélés positivement avec le nombre combiné de bourgeons et de fleurs de P. coccinea. De plus, le comportement protecteur des fourmis contre les termites était plus important pour les fleurs que les feuilles. Finalement, la production de graines était presque le double dans les fruits provenant de fleurs visitées par les fourmis en comparaison avec les fleurs desquelles les fourmis avaient été artificiellement exclues. Nos résultats suggèrent que les fourmis jouent un rôle protecteur pour les fleurs de P. coccinea contre les herbivores et les voleurs de nectar augmentant ainsi le succès reproducteur des plantes. Mots-clés : forêt amazonienne, interactions fourmi-plante, nectaires extrafloraux, protection des fleurs, voleurs de nectar, valeur adaptative des plantes. Nomenclature: Ribeiro et al., 1999.



Introduction

Extrafloral nectaries (EFNs) are specialized nectarsecreting glands that are not directly involved in plant pollination (Elias, 1983), although a few EFNs are used to attract pollinators secondarily (e.g., Poinsettia; Bentley, 1977). They are notably variable in terms of both structure and location, and are a common feature among tropical angiosperms (see Bentley, 1977 and Elias, 1983 for a review). In the last three 1Rec.

2005-08-09; acc. 2006-02-06. Associate Editor: Roy Turkington. 2Author for correspondence.

decades, a series of studies have addressed possible ecological functions of EFNs, particularly the idea that these nectaries attract ants that, in turn, protect plants against herbivores (Bentley, 1977). Although some studies did not find evidence of ant protection (O’Dowd & Catchpole, 1983; Rashbrook, Compton & Lawton, 1992), the protective role of ants has been demonstrated to translate into increased plant fitness via the deterrence of (1) leaf herbivores (Koptur, 1979; Smiley, 1985); (2) bud or flower herbivores (Schemske, 1980; Rico-Gray & Thien, 1989; Oliveira et al., 1999); and (3) seed predators (Keeler, 1981; Barton, 1986).

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Leal et al.: Ant protection in Passiflora coccinea flower

In addition to these interactions, it has been hypothesized that ant protection of plants may also function via the repellence of floral nectar thieves. This so called “nectarthief protection hypothesis” (van der Pijl, 1954) suggests that the presence of ants on EFNs of the outer floral parts (e.g., bracts) improve pollination success by displacing floral nectar thieves, thus increasing resource availability (e.g., nectar, pollen) to effective pollinators. However, empirical evidence on this process is scarce and inconsistent. For example, EFN-visiting ants on Thunbergia grandiflora (Acanthaceae) have been reported to protect flowers against nectar-thieving Xylocopa bees (van der Pijl, 1954; Faegri & van der Pijl, 1979), but this was not confirmed by Fiala et al. (1996). Furthermore, flower-visiting ants may even impose a cost to the host plant as they may not only repel the nectar thieves, but also the effective pollinators (Janzen, 1977; Willmer & Stone, 1997). Therefore, it is important not only to investigate the fitness benefits of plants resulting from ant protection against nectar-thieves, but also to understand to what extent the gained benefit is modeled by varying EFN-visiting ant communities. In mutualistic ant-plant associations composed of a large number of opportunistic ant species, for example, the overall benefit for host plants is expected to result from both the identity and the relative abundance of each ant species (Koptur, 1984; Horvitz & Schemske, 1990). Unfortunately, most studies addressing ant protection in EFN-bearing plants have focused on the protective role of particular ant species rather than entire ant assemblages (see Schemske, 1982; Rico-Gray & Thien, 1989; Oliveira et al., 1999). Thus, we still lack generalizations about both the effectiveness of ant assemblages as flower protectors and the resulting benefits for plant fitness. In this study, we assessed the assemblage of EFN-visiting ants in the neotropical woody vine Passiflora coccinea and investigated the potential fitness benefits it derives from ant activity against herbivores and floral nectar thieves. For this, we monitored the taxonomic composition, abundance, frequency, and aggressiveness of the EFN-associated ant species. To study the protective role of the observed ant assemblage, we analyzed the relationships between both EFN presence and abundance and ant defence behaviour against (1) simulated herbivores and (2) natural nectarthieving butterflies. Finally, we verified if the presence of ants on inflorescences increases both the fruit and seed set.

Methods Study site and species The study consisted of two parts and was performed in two Amazonian terra firme forest sites as follows. In the first part (August and September 1993), we investigated the EFN-visiting ant assemblage, the anti-herbivore activity, and the nectar-thief protection at forest edges along the Manaus-Caracaraí road (Br 174, km 60–62), Central Amazon, Brazil (2° 35' s , 60° 2' w ). At this site, mean annual temperature is 26.7 °C and the 30-y average of annual rainfall is 2.2 m, with a rainy season from January to May (Gascon & Bierregaard, 2001). The most abundant families are the Burseraceae, Sapotaceae, Leguminosae, and 432

Lecythidaceae (Prance, 1990). The second part of the study consisted of an exclusion experiment that was carried out in order to investigate the effects of ant protection on fruit and seed production. The experiment was performed during January and March 2000 at the Paracou Research Station in French Guiana. In this site we found a Passiflora coccinea system similar to that found on the Manaus-Caracaraí road. Paracou Research Station (5° 18' n , 52° 53' w) receives an average of 3.1 m of annual rainfall, which is concentrated around April, June, and December (Forget, Mercier & Collinet, 1999). The main families in the lowland moist forest of Paracou are Lecythidaceae, Chrysobalanaceae, and Leguminosae (Forget, Mercier & Collinet, 1999). Passiflora coccinea is a Neotropical woody vine, occurring from Central America to the south of the Amazon region. This vine inhabits forests and savannas and commonly grows on trees and shrubs in disturbed habitats, such as forest edges and treefall gaps (Benson, Brown & Gilbert, 1975; Ribeiro et al., 1999). Passiflora coccinea bears one or two pairs of EFNs on the leaf blade, near the petiole insertion, and a set of eight to twelve glands on the border of the flower bracts. It is known as a hummingbirdpollinated species (Faegri & van der Pijl, 1979), and in the case of the Manaus region effective pollination is restricted to Phaethornis superciliosus (Trochilidae) (Fischer & Leal, 2006). According to this previous study, flower visitation rates of P. superciliosus are positively correlated with the nectar volume of single P. coccinea flowers and with the resulting pollen loads on their stigmas (Fischer & Leal, 2006). Several species of butterfly were also observed visiting P. coccinea flowers for nectar consumption, but they did not contact the reproductive structures, thus acting as nectar thieves (Fischer & Leal, 2006, but see Benson, Brown & Gilbert, 1975). Ants visiting the EFNs on the bracts of P. coccinea chased butterflies as they landed on the corolla, but did not affect nectar probing by hummingbirds, perhaps due to their hovering behaviour (Fischer & Leal, 2006). Moreover, ants are not able to access the floral nectar because the nectar chamber of P. coccinea flowers is protected by a corona with several series of tied filaments (Fischer & Leal, 2006). EFN-visiting ant assemblage, anti-herbivore activity, and nectar-thief protection As Passiflora coccinea individuals are difficult to distinguish, we used clusters of flowering individuals as experimental units. Twenty focal patches were marked along a forest edge at least 10 m apart from each other to avoid patches being visited by the same ant colonies (cf. Romero & Jaffe, 1989). To describe the EFN-visiting ant assemblage, all ants on the bracts and leaves of each P. coccinea patch were recorded based on sampling periods of 30 s. As the replacement of ant species visiting the EFNs is expected to occur throughout the day (see Oliveira & Brandão, 1991), this procedure was repeated six times at 0700, 0900, 1100, 1300, 1500, and 1700 on a single observation day, totalling 120 counts (i.e., six 30-s periods in 20 patches). The frequency of each species was expressed as the total number of patches on which it was recorded during all six sampling periods (n = 120 counts). Likewise, ant abundance was the total number of individuals of each ant species recorded at

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all sampling periods (n = 120 counts). As P. coccinea bracteal EFNs can be accessed by ants in buds and open flowers we conducted a single census of both in the 20 P. coccinea patches in order to determine whether more EFNs attract more ants. Ant specimens were deposited at the insect collection of the Laboratório de Ecologia Vegetal, Universidade Federal de Pernambuco. Ant aggressiveness was determined by observing the first five interactions between two ant workers of different species on each of the 20 focal patches, resulting in a total of 100 interactions (i.e., five per patch). Observations were made in the early morning during a single day. A species was considered to be dominant if it initiated an attack or if its presence elicited avoidance behaviour during the encounter with another species (cf. Fellers, 1987). Ant aggressiveness thus referred to the percentage of the encounters in which the target species was considered dominant. To investigate the effects of EFN presence on the antiherbivory activity of ants, we used the same 20 P. coccinea patches (i.e., plants with EFNs) and selected 20 control patches composed by different plants species without EFNs and honeydew-producing homopterans. Each control consisted of the nearest similar-sized plant individual at comparable phenological stage. This method was previously used by Oliveira, Silva, and Martins (1987). The influence of EFN abundance was assessed by measuring ant activity at EFNs located on bracts (aggregated offer) and leaves (sparse offer). Ant activity was measured by using live termites as simulated herbivores, as adopted by Jeanne (1979). Two workers of Nasutitermes sp. were glued to the plant by the dorsum, one on the middle of a bract (or another outer part of flower in control patches) and the other on a green leaf. Over the next 15 min, we monitored the ants’ behaviour in response to the termite-baits and measured the elapsed time until the first attacks. This procedure was repeated simultaneously at each of the 20 control/treatment pairs, resulting in a total of 80 offered termite-baits. We also registered all interactions between EFN-visiting ants and nectar-thief butterflies on five focal flowers of P. coccinea throughout their entire anthesis period (0500-1130), a total of ca 30 h of observation. Fruit and seed production To determine if ant activity on EFNs increases the rate of fruits produced and/or the number of seeds per fruit (i.e., plant fitness), we marked 47 branches of 36 P. coccinea patches. From each branch a pair of flower buds was selected. One bud was provided with a barrier of sticky resin (Tanglefoot®, The Tanglefoot Company, Grand Rapids, Michigan, USA) to experimentally exclude ants, and the other one remained freely accessible to the ants (control). These 47 bud pairs were visited at 2-d intervals over 3 months to check the effectiveness of the resin-barrier against ants. After this period, we recorded all fruits produced and counted the number of seeds per fruit. Statistical analysis The relationships between the number of buds plus flowers of P. coccinea and the number of both ant species and individuals were analyzed by Spearman rank correlation coefficients. The difference in the average duration of

ant attacks against termites on bracts versus on leaves was analyzed using a Wilcoxon test. We also used Spearman rank correlation coefficients to analyze the relationships among the individual frequency, abundance, and aggressiveness of ant species versus the number of termites and butterflies attacked by each ant species. Here, individual ant species and their respective scores were used as replicates. The difference between the average seed number per fruit in ant-visited flowers versus ant-free flowers was compared using a one-tailed unpaired t-test as none of the bud pairs remained complete until the end of the exclusion experiment. Finally, differences among frequencies were analyzed by Chi-square tests or G-tests according to sample size. All tests mentioned here are described in Sokal and Rohlf (1995), and all analyses were performed in Systat 6.0 (Wilkinson, 1996).

Results EFN-visiting ant assemblage, anti-herbivore activity, and nectar-thief protection Twenty-two ant species of four subfamilies were recorded visiting the EFNs of P. coccinea along the Manaus-Caracaraí road (Table I). Species of Formicinae and Myrmicinae were predominant in terms of both frequency and abundance. For example, Camponotus and Crematogaster species together accounted for 81% (n = 1350) Table I. Ant species visiting the extrafloral nectaries of Passiflora coccinea in a lowland terra firme forest of the Central Amazon, Brazil. Frequencya n (%)

Abundanceb n (%)

Formicinae Brachymyrmex sp. 1 2 (1.67) Brachymyrmex sp. 2 4 (3.33) Brachymyrmex sp. 3 2 (1.67) Camponotus leydgi 13 (10.83) Camponotus lespesii 2 (1.67) Camponotus aff. blandus 64 (53.33) Camponotus sp. 1 17 (14.17) Camponotus sp. 2 12 (10.00) Camponotus sp. 3 8 (6.67)

3 (0.16) 10 (0.52) 4 (0.21) 22 (1.15) 3 (0.16) 432 (22.60) 80 (4.19) 28 (1.46) 30 (1.60)

Myrmicinae Cephalotes pusilus 2 (1.67) Crematogaster sp. 1 44 (36.67) Crematogaster sp. 2 17 (14.17) Pheidole sp. 10 (8.33)

20 (1.05) 694 (36.32) 261 (13.66) 55 (2.88)

Ponerinae Ectatomma lugens 26 (21.67) Ectatomma tuberculatum 11 (9.17) Pachycondyla villosa 4 (3.33)

87 (4.55) 61 (3.19) 5 (0.26)

Pseudomyrmecinae Pseudomyrmex oculatus 5 (4.17) Pseudomyrmex termitarius 43 (35.83) Pseudomyrmex gracilis group 12 (10.00) Pseudomyrmex sp. 1 5 (4.17) Pseudomyrmex sp. 2 5 (4.17) Pseudomyrmex sp. 3 4 (3.33)

6 74 16 11 5 4

Ant species

(0.31) (3.87) (0.84) (0.57) (0.26) (0.21)

aFrequency

is expressed as the counts of an ant species during six censuses of 20 patches (i.e., 120 counts corresponding to 100% ). bAbundance is expressed as the number of individuals of an ant species during six censuses of 20 plants.

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Leal et al.: Ant protection in Passiflora coccinea flower

of all records. Camponotus aff. blandus and Crematogaster sp. 1 were the most frequent and abundant visitors to the P. coccinea EFNs, respectively (Table I). Both the number of ant species and the number of ant individuals were positively correlated with the combined number of buds and flowers recorded at the 20 P. coccinea patches (rs = 0.72, n = 20, P