conditional outcomes in ant–plant–herbivore interactions influenced by sequential flowering

Accepted Manuscript

Title: Conditional outcomes in ant-plant-herbivore interactionsinfluenced by sequential flowering

Author: Andrea Andrade Vilela Helena MauraTorezan-Silingardi Kleber Del-Claro

PII: S0367-2530(14)00048-6DOI: http://dx.doi.org/doi:10.1016/j.flora.2014.04.004Reference: FLORA 50770

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Received date: 13-10-2013Accepted date: 12-4-2014

Please cite this article as: Vilela, Andrea Andrade, Torezan-Silingardi, Helena Maura,Del-Claro, Kleber, Conditional outcomes in ant-plant-herbivore interactions influencedby sequential flowering.Flora http://dx.doi.org/10.1016/j.flora.2014.04.004

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http://dx.doi.org/doi:10.1016/j.flora.2014.04.004

http://dx.doi.org/10.1016/j.flora.2014.04.004

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Conditional outcomes in ant-plant-herbivore interactions influenced by sequential flowering

Andréa Andrade Vilela1, Helena Maura Torezan-Silingardi1 and Kleber Del-Claro1*

1 Laboratório de Ecologia Comportamental e Interações, Instituto de Biologia, Universidade Federal

de Uberlândia, Uberlândia, Minas Gerais, CEP 38400-920, Brasil.

*Corresponding author: [email protected]

Edited by R. Lösch

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Abstract Mechanisms that affect a host plant’s ability to face herbivory are subjects of ongoing

interest. Plant reproductive phenology plays a key role in the dynamics of communities in many

ways. In ant-plant-herbivore interactions, host-plant phenology affects traits of its herbivores which

in turn determine what traits ants must have to benefit the host-plant. Diversity of plant

phenological traits could influence the ecological diversity of coevolved ant-plant mutualisms.

In the Brazilian savanna, members of several plant families resprout and bloom

simultaneously. However, some shrubs of the family Malpighiaceae exhibit sequential flowering,

and four of these species also present extrafloral nectaries that attract ants. Here we determine

whether their phenological patterns results in a shared herbivore guild and if this may be harmful to

these plant species, making the association with ants critical to the plant optimal development and

reproductive success. Plant phenology, herbivory, and the richness and abundance of ants and

herbivores were recorded on control (with unrestricted ant access) and treatment (ant-excluded)

shrubs. Floral-bud production and fruit set were also quantified. The plants flowered sequentially

with brief periods of overlap, benefitting the guild of generalist herbivores. A cluster analysis

indicated that 32 herbivore species were associated with these Malpighiaceae, with substantial

species overlap. In all species, ants reduced leaf-area loss but not the damage to reproductive

structures. Some floral herbivores presented adaptations to avoid or appease ants. We suggest that

plant phenology directly influences the outcomes of these ant-plant-herbivore interactions and the

related ecological networks.

Keywords: ants, cerrado, floral phenology, Malpighiaceae, savanna, multitrophic networks.

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Introduction As the base of terrestrial trophic chains, plants are consumed by a wide range of organisms,

including microbes, invertebrates and vertebrates. The pressure that herbivores exert over plant

development and fitness has led plants to develop numerous defensive strategies (Marquis, 2012;

Mortensen, 2013). While some defenses are constitutive, others are induced only upon the

perception of an attack to allow for optimal resource allocation (Karban and Baldwin, 1997;

Campbell and Kessler, 2013). One interesting plant trait to escape from herbivory is to resprout or

bloom during a season when the main herbivores are less common in the field (Coley and Barone,

1996). Some plant families produce new leaves and flowers during the same season; others produce

leaves and flowers at distinct times, such as during the dry and wet seasons (Raven, 2012).

According to Staggemeier and Morellato (2011), most tropical plant species rely upon animal

vectors for pollination and seed dispersal, and temporal and spatial variations in flowering and

fruiting phenology strongly affect animals that rely upon flowers or fruits as a food resource.

Plants present different patterns of flowering, such as aggregated, segregated (e.g. Gentry,

1974; Gotelli and Graves, 1996; Murali and Sukumar, 1994) or indistinguishable from what would

be expected by chance (e.g. Rathcke, 1984). Several evolutionary hypotheses have been proposed to

explain the distinct patterns observed in flowering times among species. Initially, Robertson (1895)

suggested that plants with the same pollinators will avoid competition through staggered flowering

and thus will increase their reproductive success. Another hypothesis states that staggered flowering

could be a result of interaction between early-flowering and later-flowering species, wherein the

first-flowering species facilitates the pollinators of the next species to flower (Waser and Real,

1979). The analysis of flowering strategies is a complex issue because they are the result of an

interacting set of abiotic factors, plant traits and plant-animal interactions (Armbruster, 1995). In a

consumer-resource perspective, sequential flowering may represent a plant defensive strategy

against floral herbivores (Coley and Barone, 1996; Marquis and Lill, 2010).

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Biotic defenses, like the association with predators, most commonly ants, may also represent

an important defensive strategy against action of herbivores in terrestrial ecosystems. Ant-plant

relationships have made enormous contributions to our understanding of tropical communities

(Rico-Gray and Oliveira, 2007; Rosumek et al., 2009), and these interactions are particularly

pervasive in the Cerrado (the Brazilian tropical savanna) due to the high incidence of insect- and

plant-derived exudates on foliage, which promotes intense ant activity on the vegetation (Rico-Gray

and Oliveira, 2007, and references therein). The Cerrado is the most diverse tropical savanna in

fauna and flora, and one remarkable characteristic of this ecosystem is its great seasonal variation

(Oliveira-Filho and Ratter, 2002). The reproductive success of individual plant populations often

depends on flowering phenology allowing plants to take advantage of temporally periods of

favorable conditions (Mahoro, 2002). Studies comparing conditional outcomes in ant-plant-

herbivore interactions mediated by temporal variation in host-plant phenology are of great relevance

to the ecology of interactions and the conservation of natural communities. However, such studies

are rare, especially in tropical America (Rosumek et al., 2009).

Experimental ant-exclusion studies have shown that the absence of ants can increase leaf-

area loss in plants possessing extrafloral nectaries (Oliveira, 1997; Korndörfer and Del-Claro,

2006). The absence of ants can also reduce fruit set (Oliveira et al., 1999; Nascimento and Del-

Claro, 2010), seed production (Vesprini et al., 2003) and viability (Sobrinho et al., 2002). However,

data concerning the effectiveness of ant–plant mutualisms are sometimes controversial (see, e.g.

Holland et al., 2011; Nahas et al., 2012). Nevertheless, despite their importance to the study of

mutualisms and to the structure and maintenance of natural communities (e.g., Rico-Gray and

Oliveira, 2007; Bronstein, 2012), ant-plant interactions are most often studied in single plant species

over a limited time period (Rosumek et al., 2009), ignoring sequential events. Recently, some

researchers have shown the impact of ant-plant interactions on communities using diverse

ecological network approaches (e.g., Blüthgen et al., 2007; Ings et al., 2009; Lange et al., 2013, and

references therein) and have focused on the ecological factors that may cause a mutualism to vary in

space and time, resulting in "conditional mutualism" (Herre et al., 1999; Del-Claro and Oliveira,

2000; Alves-Silva and Del-Claro, 2013).

In the Cerrado of central Brazil, shrubs of the family Malpighiaceae are diverse and

abundant (Anderson, 1990; Gates, 1982). Several species exhibit sequential phenological

development, in which individuals of different species resprout, bloom and set fruit sequentially

over time (Barros, 1992; Costa et al., 2006; Torezan-Silingardi, 2007; Mendes et al., 2011). These

species also possess extrafloral nectaries (EFNs) and have close relationships with protective ants

(Torezan-Silingardi, 2011; Alves-Silva et al., 2013). In Cerrado, the ant-plant-herbivore interactions

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occur within multitrophic systems whose outcomes are strongly influenced by plant phenology

(Lange et al., 2013). Here we used a set of Malpigiaceae-ant-herbivore associations as model to

determine the following: a) whether herbivores shift among related host-plant species over time in

response to variation in plant phenology; and b) whether the outcomes of ant-plant interactions vary

depending upon the associated herbivores and ants. Our major hypotheses are that the sequential

flowering of related host plants results in a shared herbivore guild over time that may be quite

harmful to these plant species, making the association with ants critical to the plants reproductive

success.

Materials and methods

Study site and plant species Fieldwork was carried out from May 2008 to June 2009 at the Reserva Ecológica do Clube

Caça e Pesca Itororó de Uberlândia (CCPIU; 18°59’S, 48°18’W), Uberlândia, Minas Gerais State,

Brazil. We used a 400-ha Cerrado site consisting of a dense scrub of shrubs and trees, known as

Cerrado sensu stricto (Oliveira-Filho and Ratter, 2002). The climate is markedly seasonal, with a

dry winter (April to September) and a rainy summer (October to March) – for additional details, see

Réu and Del-Claro, 2005).

Four Malpighiaceae species were selected for the study: Peixotoa tomentosa A. Juss.,

Banisteriopsis laevifolia (A. Juss) B. Gates, Banisteriopsis campestris (A. Juss.) Little, and

Banisteriopsis malifolia (Ness and Mart.) B. Gates. These species were chosen because they are

small shrubs with paired EFNs on the leaf base and are common in the Cerrado and abundant at the

study site (Torezan-Silingardi, 2007).

Experiments During the month in which each species sprouted (when the shrubs bore vegetative buds but

not leaves), we tagged 30 individuals of similar size and architecture (1–2 m tall, with 5–7 stems)

that were at least 3 m apart. Thus, the ant-exclusion experiments began in May 2008 for P.

tomentosa, July 2008 for B. laevifolia, November 2008 for B. campestris and February 2009 for B.

malifolia. For each species, by the flip of a coin, we designated tagged shrubs as control plants

(with unrestricted ant access; N=15) or treatment plants (with ants excluded by applying

Tanglefoot® resin on the main plant stem; N=15). In treatment plants, ants were manually removed

and the trunk was covered with a 5 cm broad adhesive paper strap to which a layer of sticky resin

was applied. We removed surrounding vegetation that ants could use as bridges to gain access to

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these plants. To be sure that the sticky resin did not interfere in the results, in control plants we also

covered the trunk with a paper strap, but we applied the resin only on one side of the trunk.

Data Collection Each plant was monitored every two weeks during its four-month reproductive period. The

plants were always inspected by the same observer, and the richness and abundance of herbivores

on the reproductive and non-reproductive plant parts were recorded. We quantified herbivory (leaf

area loss) monthly in the first week of each month. To determine the mean monthly herbivory, we

recorded data from nine leaves per plant, three from the most apical stem, three from a middle stem

and three from the most basal stem. This procedure was done without leaf removal. Measurements

of herbivory rates on leaves were assessed by placing leaves on a transparent grid (divided into

millimeters). An index of herbivory from each leaf was calculated as the proportion of points in the

grid falling within damaged and undamaged areas of the leaf blade (Moreira and Del-Claro, 2005).

Additionally, data about behavior of the animals at the time of observation were noted, including

their topical position (on leaves, stems, buds, or flowers or feeding on EFNs). An animal was

considered an herbivore if it was observed sucking, chewing, perforating or grasping the plant

tissue. Voucher specimens were collected from non-experimental plants for identification. Plant

phenology (developmental stage of leaves, buds, flowers and fruits) was recorded by A. Vilela,

following the practice of Torezan-Silingardi and Oliveira (2004) during May 2008 to June 2009.

Statistical analysis Circular statistical analyses were made using the following phenological variables: date of

first bud, first flowering and first fruiting, date of peak of bud, flowering and fruiting. To calculate

the circular statistic parameters, months were converted to angles from 0 = January to 345 =

December at intervals of 15 because the measurements were made every two weeks (Cardoso, et al.,

2012). The frequency of individuals at each phenological phase for all species (n = 4) was

considered for calculating the parameters: the mean vector (µ), length of mean vector (r), median,

circular standard deviation, Rayleigh test z and Rayleigh test p. The mean date for each phenophase

is determined by converting the mean angular directions to corresponding mean dates (see

Morellato et al., 2000, 2010; Staggemeier et al., 2010). The phenological data were analyzed with

the statistical software Oriana 4.0.

A null model calculation was performed to indicate if the temporal overlap among species

should be less than expected by chance, which represent a segregated or sequential phenology.

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Overlap was quantified via Pianka (Pianka, 1973) and Czechanowski (Feinsinger et al., 1981)

indices and the values were generated using a randomization algorithm ('Rosario'). This algorithm

was designed specifically for use with important interval data (Castro-Arellano et al., 2010).

'Rosario' maintains the shape of the empirical activity distributions for each species in the randomly

generated matrices by shifting entire activity patterns over a random number of intervals. For each

analysis, overlap indices were calculated for 10,000 randomly generated matrices of temporal

flowering phenology, creating a null distribution of overlap values. Then a one-tailed test was

conducted and the p-value was calculated as the proportion of randomizations that resulted in

overlap that is equal to or less than the empirical overlap value (observed) – see, e.g., Brito et al.

(2012). Simulations were conducted with the TimeOverlap program (Castro-Arellano et al., 2010).

The similarity of herbivore composition among plant species was evaluated using a cluster

analysis (Freitas et al., 2002). An incidence matrix was constructed with the presence or absence of

each herbivore species on each plant species. A dendrogram was then constructed using the Jaccard

index and single linkage with the software Systat 12 (Jurzenski et al., 2012). Differences in leaf-

area loss were compared using repeated-measures ANOVA after arcsin transformation of the

percentage values. The fruit set and herbivore abundance were compared using the Mann-Whitney

U test because the data were not normally distributed even after transformation.

Results In the circular diagrams the mean angles of onset and peak for all phenophases were not

significantly seasonal (Rayleigh test P > 0.05) and the lengths of mean vectors (r) were nearly 0

(Table 1). The vector r varies from 0 (when phenological activity is distributed uniformly

throughout the year) to 1 (when phenological activity is concentrated around one single date or

mean angle), cf. Zar (1996). Thus, the results showed that phenological activity of the

Malpighiaceae species studied is distributed uniformly throughout the year featuring a segregated

phenology.

The null model simulations indicated that the distributions overlap was significantly less

than expected by chance, which means that species exhibited sequential flowering with little

overlap in their reproductive periods (Pianka = 0.202, P < 0.001; Czechanowski = 0.968, P <

0.001). The first to blossom was P. tomentosa in June 2008, followed by B. laevifolia, B. campestris

and B. malifolia (Fig. 1).

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Cluster analysis showed the greatest similarity in herbivore composition between P.

tomentosa and B. laevifolia (70%). Between these two species and B. campestris the similarity was

almost 65%. Banisteriopsis malifolia shared almost 50% of its herbivores with the other plant

species (Fig. 2).

The presence of ants significantly reduced the leaf damage caused by herbivores in all plants

(P. tomentosa: F = 616.8, P < 0.001, DF = 1; B. laevifolia: F = 604.8, P < 0.001, DF = 1; B.

campestris: F = 603.6, P < 0.001, DF = 1; B. malifolia: F = 544, P < 0.001, DF = 1; Fig. 3) but did

not affect fruit set (P. tomentosa: U = 49, P = 0.67, n = 30; B. laevifolia: U = 97, P = 0.52, n = 30;

B. campestris: U = 101, P = 0.61, n = 30; B. malifolia: U = 89.5, P = 0.34; n = 30). In this plant-

herbivore system, each plant species had a main group of more abundant herbivores (mostly floral

herbivores) and a secondary group of less abundant herbivores (secondary herbivores). The main

herbivores of each plant species caused considerable damage to their respective hosts, mainly

injuring the reproductive structures and reducing the benefit of ants to the plants’ fitness (fruit set).

Thrips (Thysanoptera: Heterothrips peixotoa) were the most numerous herbivores on P.

tomentosa during the plant reproductive season (n = 587; Fig. 4), far outnumbering orthopterans (n

= 12), hemipterans (n = 11), coleopterans (n = 10) and lepidopteran larvae (n = 8). Thysanopterans

infested control plants and experimental plants equally (U = 85, P = 0.26, n = 30), while secondary

herbivores were significantly more abundant on ant-excluded plants (U = 63, P = 0.04, n = 30; Fig.

5). Thus, ants effectively removed or chased away herbivores from P. tomentosa, except thrips.

Ectatomma tuberculatum was the most common visiting ant species on P. tomentosa.

Curculionid beetles (Curculionidae: Anthonomus sp.) were the most numerous herbivores on

B. laevifolia (n = 3906; Fig. 6), followed by thysanopterans (n = 161), hemipterans (n = 76), other

coleopterans (n = 15) and orthopterans (n = 9). Curculionid larvae are endophytic herbivores that

infest the buds and flowers, eating the plant embryonic tissue (Torezan-Silingardi, 2011). These

beetles infested control plants and ant-excluded plants equally (U = 102, P = 0.66, n = 30), while

the other herbivores were significantly more abundant on ant-excluded plants (U = 59.5, P = 0.03, n

= 30; Fig. 5). Thus, ants effectively removed exophytic herbivores but not endophytic larvae from

these plants. Cephalotes sp. was the most common visiting ant species on B. laevifolia.

Mealybugs (Hemiptera: Coccoidea) were the main floral herbivores on B. campestris (n =

478) and B. malifolia (n=386; Fig. 7). These mealybugs are trophobiont insects, attracting and

feeding attending ants while sucking at the base of the floral pedicel. In B. campestris, the

secondary herbivores were thysanopterans (n = 133), coleopterans (n = 51), hemipterans (n = 46)

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and orthopterans (n = 28). In B. malifolia, the secondary herbivores were thysanopterans (n = 112),

orthopterans (n = 98), hemipterans (n = 86), coleopterans (n = 32) and lepidopteran larvae (n = 2).

Mealybugs were significantly more abundant on control plants of both B. campestris (U = 47, P <

0.05, n = 30) and B. malifolia (U = 65, P < 0.05, n = 30), while secondary herbivores were more

abundant on ant-excluded plants of B. campestris (U = 60, P < 0.05, n = 30; Fig. 8) and B. malifolia

(U = 42, P < 0.05, n = 30; Fig. 8). Crematogaster sp. and Camponotus sp. were the most common

visiting ant species on B. campestris and B. malifolia, respectively.

Discussion The hypothesis was confirmed that the sequential flowering of related host plants results in a

shared herbivore guild over time that may be quite harmful to these plant species. The sequential

flowering of the studied species of Malpighiaceae favors the use of these plants by a similar

herbivore guild over time. The interactions with ants were important to plants to reduce the

abundance of sharing herbivores (fig. 5 and fig. 8) and foliar herbivory (fig. 3). The strength of

positive effects on reproductive structures was affected by the variation in the morphological and

behavioral characteristics of certain herbivore groups and ants associated with particular host plants.

Thus, the association with ants is critical to the optimal development of these plant species that

present sequential flowering in the Brazilian tropical savanna.

Sequential flowering can be initiated by seasonal changes in rainfall, temperature and gain

of solar radiation (Rathcke and Lacey, 1985). Plants with sequential flowering, such as the shrubs

studied here, succeed each other in producing floral rewards throughout the year, generating the

conditions needed to maintain pollinators and other floral visitors (Gentry, 1974; Newstron et al.,

1994; for examples in Malpighiaceae see Sigrist and Sazima, 2004; Costa et al., 2006). Our results

show that the sequential resprouting and flowering of Malpighiaceae species in the Cerrado also

provides an uninterrupted food supply to a diverse herbivore guild. In Malpighiaceae, immature

structures (e.g., young leaves, buds and flowers) generally have low structural resistance to physical

damage, making the shoots and inflorescences especially attractive to chewing and sucking insects

(Del-Claro et al., 1997). This fact, associated with the close resemblance in floral structure among

Malpighiaceae species (Anderson, 1979), may contribute to the observed similarity of the herbivore

fauna.

The shared herbivore guild may be quite harmful to these members of Malpighiaceae,

making the association with ants decisive for optimal development of these plants. Our

experimental manipulative study showed that the protection provided by ants is significantly biased

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against foliar herbivory, as observed in other Cerrado plants (Costa et al., 1992; Del-Claro et al.,

1996; Oliveira, 1997). However, these benefits do not extend to the reproductive structures. The

lack of effective ant protection for the plants’ reproductive structures can be explained by seasonal

and temporal variation (Alves-Silva and Del-Claro, 2013). Temporal variations in the abundance of

the main plant herbivores and behavioral characteristics of the herbivores and ants may reduce or

eliminate the beneficial effects of ants on plant productivity. On the other hand, the significant

reduction in leaf area loss due ant presence may improve plant survivorship and future reproductive

success (Marquis, 2012). Thus, our results corroborate the hypothesis that the outcomes of ant-plant

interactions depend upon the ecological and behavioral features of associated ant and herbivore

species (Del-Claro and Oliveira, 2000).

The peak abundance of thysanopteran insects coincided with the reproductive period of

Peixotoa tomentosa. This group of herbivores consists of small phytophagous and mycophagous

insects (Mound and Kibby, 1998) that develop largely in floral chambers (Del-Claro et al., 1997;

Alves-Silva and Del-Claro, 2013) and consume developing flowers, pollen and fruits (Del-Claro

and Mound, 1996; Pinent et al., 2005). These insects are by far the main herbivores of P. tomentosa.

In addition to the morphological characteristics of thrips, we considered certain morphological

characteristics of the ants associated with the plants. Inside the floral chambers, thrips can be

captured only by minute ants, such as Crematogaster/Myrmicinae and Brachymyrmex/Formicinae

(Del-Claro et al., 1997). The ants observed during the flowering season of P. tomentosa were

mainly Ectatomma (Ectatomminae) and Camponotus (Formicinae) species, which have

morphological constraints (larger size) that prevent them from entering the floral chambers.

Therefore, the ants that interact with P. tomentosa are ineffective in removing and reducing the

abundance of thrips, allowing severe damage to the plants’ reproductive structures regardless of the

presence of ants, as observed by Del-Claro et al. (1997). This relationship between the

morphological limitations of certain ant genera and the inefficient removal of thysanopteran

herbivores indicates that the identity of the ant species present in the field during the P. tomentosa

reproductive season may be more critical to the outcome of the ant-plant interaction than the high

abundance of thrips.

The Banisteriopsis laevifolia shrubs may have been strongly affected by the high abundance

of their main herbivore. The peak abundance of beetles of the genus Anthonomus (Anthonominae:

Curculionidae) coincided with the flowering period of B. laevifolia, and the ants were not

significantly effective in removing these beetles. These herbivores feed and complete their

development inside the flower buds. Larval development within plant structures benefits beetles by

ensuring a steady food supply, protection against sun and drying winds and shelter against predators

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and parasites (Clark and Martin, 1987; Torezan-Silingardi, 2011). Therefore, the high abundance of

curculionids during the reproductive period of B. laevifolia most likely causes extensive damage to

the buds of this species, which may not be intensely or fully protected by the associated ants. In a

previous study by Torezan-Silingardi (2007), beetles of the genus Anthonomus (Anthonominae:

Curculionidae) had the greatest species richness (eight species), but their low abundance mitigated

the damage to their plant hosts.

In this study, mealybugs were the major herbivores on Banisteriopsis campestris and B.

malifolia and were present mainly on plants with ants. Del-Claro and Oliveira (2000) have shown

that the benefits to the host plant in plant-ant-hemipteran interactions vary depending upon the

behavior of the associated herbivores and ants. Here, the association of ants with coccoid herbivores

most likely provides benefits for the plant leaves, as observed in similar systems (Moreira and Del-

Claro, 2005). However, the high abundance of these animals during the reproductive periods of B.

campestris and B. malifolia negatively affects these plants’ reproductive success.

The studied system shows that the interactions between ants, plants and their herbivores occur

within multitrophic networks whose dynamics, which determine the outcomes of the interactions,

are strongly influenced by the sequential flowering of plants (e.g. Lange et al., 2013). In addition to

various bottom-up effects, the phenological synchronization between herbivores and their host

plants frequently determines the quantity and quality of food resources and abundance of

herbivores, directly impacting populations and communities (Kerslake and Hartley, 1997; Yukawa,

2000). Unfortunately, few entomologists record detailed phenological data on host plants, and little

information is available linking sequential resprouting and/or flowering with the associated

herbivore fauna. We hope that these conditions will change in the near future because accumulation

of phenological data is necessary also to assess the effects of global warming on the

synchronization of herbivores with host-plant phenology (e.g. Yukawa, 2000).

Acknowledgements We thank C. Belchior, D. Lange, L. Nahas, Robert J. Marquis, S. Sendoya, R. Lösch and

two anonymous reviewers for reviewing the manuscript. We also thank CNPq for funding this

research (KDC/HMTS - 476074; 472046; 301248) and CAPES and FAPEMIG for awarding

fellowships to AAV.

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Figure captions

Fig.1 Linear graphic with the intensity of flowering of Malpighiaceae species showing the sequential flowering of Peixotoa tomentosa, Banisteriopsis laevifolia, B. campestris and B. malifolia, respectively, in a Brazilian tropical savanna between June of 2008 and 2009 (axis and mean intensity of flowering phenology being 1 = 25% and 2 = 75%; n = 30)

Fig. 2 Cluster analysis of the guild of herbivores associated to four Malpighiaceae species

(Peixotoa tomentosa, Banisteriopsis laevifolia, B. campestris and B. malifolia) in a Brazilian

tropical savanna between June of 2008 and 2009 (Jaccard index).

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Fig. 3 Comparative analysis of herbivory (leaf area loss; median ± 1SE) between control (white

bars – ant access) and treatment (gray bars – ant exclusion) shrubs of four Malpighiaceae species

(Peixotoa tomentosa, Banisteriopsis laevifolia, B. campestris and B. malifolia) in a Brazilian

tropical savanna. The asterisks (**) means P < 0.001 (Repeated Measures ANOVA).

Fig. 4 Circular scatterplots for the occurrence of: plants flowering; stems per plant with active

extrafloral nectaries (NEF); plants visited by ants; trips abundance on plants of Peixotoa

tomentosa (n = 30), in a Brazilian tropical savanna between June and September of 2008. The

straight line between bars indicates the circular median with standard deviation (95% CI). The

four histograms show marked seasonality (P < 0.001, Rayleigh Z-test).

Fig. 5 Comparative analysis of abundance of secondary herbivores (median ± 1 SE) between

control (white bars – ant access) and treatment (gray bars – ant exclusion) shrubs of Peixotoa

tomentosa and Banisteriopsis laevifolia in a Brazilian tropical savanna. The asterisks (**) means

P < 0.05 (non-parametric test, Mann Whitney U-test).

Fig. 6 Circular scatterplots for the occurrence of: plants flowering; stems per plant with active

extrafloral nectaries (NEF); plants visited by ants; Curculionidae abundance; on plants of

Banisteriopsis laevifolia (n = 30), in a Brazilian tropical savanna between August and November

of 2008. The straight line between bars indicates the circular median with standard deviation

(95% CI). The four histograms show marked seasonality (P < 0.001, Rayleigh Z-test).

Fig. 7 Circular scatterplots for the occurrence of: plants flowering; stems per plants with active

extrafloral nectaries (NEF); plants visited by ants and mealybugs abundance on plants of

Banisteriopsis campestris (n=30) and Banisteriopsis malifolia (n = 30) in a Brazilian tropical

savanna between December of 2008 and March of 2009 and March 2009 until June of 2009,

respectively. The straight line between bars indicates the circular median with standard deviation

(95% CI). The four histograms show marked seasonality (P < 0.001, Rayleigh Z-test).

Fig. 8 Comparative analysis of abundance of secondary herbivores and mealybugs (median ± 1

SE) between control (white bars – ant access) and treatment (gray bars – ant exclusion) shrubs of

Banisteriopsis campestris and Banisteriopsis malifolia in a Brazilian tropical savanna. The

asterisks (**) means P < 0.05 (non-parametric test, Mann Whitney U-test).

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Table 1 Circular statistical analysis testing for seasonality of phenological patterns of four

Malpighiaceae species in a Brazilian tropical savanna between June of 2008 and 2009. Rayleigh test

were performed for significance of the mean angle, and the mean data were omitted because they

were not significant.

Flower bud Flower Fruit Onset Peak Onset Peak Onset Peak

Mean data 16/07/2008 15/06/2008 02/07/2008 25/07/2008 17/07/2008 08/07/2008 Number of Observations 110 109 97 90 87 88 Mean Vector (µ) 197,599° 166,697° 183,012° 206,112° 198,797° 219,599° Length of Mean Vector (r) 0,162 0,101 0,174 0,169 0,175 0,177 Median 225° 225° 225° 240° 240° 255° Circular Standard Deviation

109,408° 122,594° 107,109° 108,082° 106,992° 106,622°

Rayleigh Test (Z) 2,87 1,12 2,945 2,563 2,661 2,758 Rayleigh Test (p) 0,06 0,32 0,06 0,08 0,07 0,07

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Figure 8

conditional outcomes in ant–plant–herbivore interactions influenced by sequential flowering

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