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Herbivory-induced overcompensation and resource-dependent production of extrafloral nectaries in Luffa cylindrica (Cucurbitaceae)
Acta Oecologica 93 (2018) 1–6
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Herbivory-induced overcompensation and resource-dependent production of extrafloral nectaries in Luffa cylindrica (Cucurbitaceae)
T
Poliana Fernandes Souza Limaa, Alberto L. Teixidoa, Elder Antônio Sousa Paivaa,∗ a
Departamento de Botânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 30161-970, Belo Horizonte, Minas Gerais, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Damage-dependent response EFN density Extrafloral nectary Leaf area Plant nutritional status Resource-dependent response
Extrafloral nectaries (EFNs) are nectar secretory structures involved in the indirect defense of plants. In the sponge gourd (Luffa cylindrica), EFNs commonly occur on the lower surface of leaf blades and stipules and remain functional until leaf senescence. To test the hypothesis that the development of EFNs is influenced by herbivore damage and resource availability, we grew Luffa cylindrica under different concentrations of Hoagland's nutrient solution (nutrient-poor conditions: 10%, 50%; and control condition: 100%) and two herbivory treatments (damaged and undamaged leaves). We collected ten leaves from treated plants to quantify leaf area and EFN density. Overall, leaf area increased and EFN decreased in damaged plants, but this significantly depended on nutritional status. In undamaged plants, EFN density tended to remain constant, whereas foliar area increased with nutrient input. Under herbivory, foliar area increased at 10% but decreased at 50 and 100% of nutrients in relation to undamaged plants, whereas EFN density tended to increase with nutrient availability to exceed undamaged plants under control concentrations. Plants under nutrient-poor conditions subjected to herbivory exhibited an increased foliar area, characterizing a compensatory mechanism. Our results suggest that herbivore-induced indirect defense is a damage- and resource-dependent response in Luffa cylindrica. These findings contribute to understanding the factors that modulate indirect defenses and plant-herbivore-environment interactions.
1. Introduction Plants have to deal with a diverse assemblage of herbivores, which can consume significant amounts of biomass and reduce plant reproductive success (Crawley, 1983; Coley and Barone, 1996; Strauss et al., 2002; Turcotte et al., 2014). Consequently, plants have developed a diversity of structures and evolutionary strategies to provide protection against herbivory. For example, some mechanisms provide indirect defense by attracting natural predators of herbivores (Agrawal, 1998; Karban and Baldwin, 2007; Yamawo, 2017). However, resource allocation to defense may compromise plant development and reproduction (Strauss and Agrawal, 1999; Schiestl et al., 2014). Thus, induced defenses stimulated at the moment of damage have been favored by resource-mediated natural selection (Zangerl and Rutledge, 1996; Ness, 2003; Meldau et al., 2012; Lortzing et al., 2016), which entail a resource cost only when strictly necessary and thus allow increased allocation of resources to other functions. Herbivore-induced defense strategies of plants are generally resource-dependent (Stamp, 2003; Mondor et al., 2006; Karban, 2011; Yamawo, 2017). Following this assumption, resource allocation
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patterns for induced defenses mostly follow optimal defense theory, which suggests that plant organs and structures with a high energetic value and a subsequent higher herbivore incidence show higher investment in defense (McKey, 1974; Mondor et al., 2006; Xu and Chen, 2015). Therefore, one could experimentally evaluate whether both resource availability and mechanical damage significantly affect the induction of defenses. Evidence in this sense would potentially explain reduced defense responses in the absence of damage and in resourcepoor conditions. In some cases, the development and reproduction of plants damaged by herbivores can be even greater than that of undamaged plants. Such a positive effect of herbivory on plant performance is referred to as a mechanism of overcompensation. Herbivore-mediated selection for overcompensatory responses favors increased fitness of damaged plants and plant traits that increase the probability of attack (Paige and Whitham, 1987; Vail, 1992; Agrawal, 2000; Poveda et al., 2010). The benefits derived from overcompensation are associated with increased lateral branching, stimulated growth and subsequent improved plant reproduction (Agrawal, 2000; Rautio et al., 2005; Sun et al., 2009; Olejniczak, 2011). Interestingly, plant compensatory responses to
Corresponding author. E-mail address: [email protected] (E.A. Sousa Paiva).
https://doi.org/10.1016/j.actao.2018.10.001 Received 23 March 2018; Received in revised form 15 August 2018; Accepted 9 October 2018 1146-609X/ © 2018 Elsevier Masson SAS. All rights reserved.
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randomly arranged in a greenhouse where they were maintained under a 12/12 hours light/dark cycle with a light intensity of 400 μmol/m−2/ s−1.
herbivory may also be affected by resource availability; however, although it is still unclear whether higher resource levels increase, or not, tolerance of herbivory, it appears to be largely dependent on the most limiting resources (see Wise and Abrahamson, 2007). Some of herbivore-induced indirect defense strategies comprise a diversity of secretory structures that excrete chemical compounds that promote predator-mediated protection against herbivory (Stamp, 2003; Heil, 2008; Nahas et al., 2012; Lortzing et al., 2016). A broad taxonomic array of species is known to produce extrafloral nectaries (hereafter, EFNs; Heil et al., 2009; Weber and Keeler, 2013; Weber et al., 2015). EFNs are structures involved in nectar secretion, a valuable energetic resource for arthropod species that reduce the presence of herbivores and other plant enemies (Heil, 2011). Therefore, EFNs are not directly linked to pollinator attraction, but rather they are commonly responsible for indirect biotic defense of plants by attracting other species such as ants and wasps, which by means of their presence and activity promote plant protection (Schmid, 1988; Byk and Del Claro, 2011; Gonçalves-Souza et al., 2016). In this regard, EFNs are key elements for increased biomass, leaf production, reproduction and fitness of plants (reviewed in Rosumek et al., 2009). EFNs are also herbivore-induced structures largely dependent on resource availability (Mondor et al., 2006; Karban, 2011; Yamawo et al., 2012, 2014), so EFN production can readily decrease along plant ontogeny due to tradeoffs with other functions or changes in its inducibility, but generally increasing under damage events (Quintero et al., 2013 and references therein). Therefore, changes in production of EFNs are essential to understand the induction of indirect defenses related to herbivore damage and resource dependence. The gourd family, Cucurbitaceae, is especially rich in EFN-bearing species, including the annual and monoecious sponge gourd (Luffa cylindrica), which possesses disc-shaped EFNs on its leaf surface, stipules, bracts, bracteoles and fruits (Agarwal and Rastogi, 2010; Nesom, 2015). These EFNs are conspicuous, numerous (reaching 80 by leaf blade) and produce nectar as droplets, mainly during the stages of leaf expansion and in young expanded leaves. Associated with this species is a highly diverse set of insect herbivores and their natural predators, in particular, ants are the most frequent group (Agarwal and Rastogi, 2010). The number of EFNs in L. cylindrica is positively correlated with predator ant patrolling time, representing a relevant indirect defense strategy (Agarwal and Rastogi, 2010). Here we aim to understand the relationship between nutritional status and defense investment by examining foliar area and EFN density of L. cylindrica cultivated under different nutrient concentrations and mechanical damages. We exclusively focused on EFN density as it is a true putative measure of defense readily comparable with previous studies showing herbivory- and resource-dependent EFN production (e.g. Mondor et al., 2006; Lortzing et al., 2016). We specifically tested if: (1) leaf area and EFN density vary with the nutritional status and simulated herbivory; and (2) EFNs density varies along plant ontogeny between the most basal and apical leaves developed after the simulated herbivory.
2.2. Nutrient solution To evaluate whether EFN density is damage- and resource-dependent, three nutrient solutions of different concentrations (10, 50 and 100% strength of Hoagland nutrient solution) were used, on 30 randomly selected seedlings. The 100% concentration was used as nutrient control group, since the plants received normal and appropriate levels of mineral nutrients, whereas the concentrations of 10 and 50% of nutrients were used as experimental treatments (reduction of resources). The volume of liquid in the pots was filled daily with deionized water to replace loss by evaporation, and the solution was renewed weekly to maintain the experimental nutrient concentrations, until the end of experimental period (80 days). 2.3. Herbivory To determine whether EFN density can be induced by mechanical damage, we simulated herbivory on 15 plants under each nutrient treatment (i.e. 10, 50 and 100%) 60 days after seedling transplantation, whereas 15 plants in each nutrient condition were unmanipulated and left as controls. All plants, regardless of treatment, were in a non-reproductive stage. Simulation of herbivory consisted of removal of 15% of the foliar area by means of a single-hole punch (Staples 10573-CC, Staples Inc, SP, Brazil). Although the amount of foliar area to be removed to simulate herbivory is somewhat variable in the literature, 15% fits the mean damage to plants in natural communities (Strauss and Agrawal, 1999) and was sufficient to test our hypotheses (see Results). Foliar area was previously assessed for 50 randomly chosen leaves from adult plants by using AxioVision SE64 (AxioVision Microscopy Software, 2009; ZEISS, Germany). We subsequently estimated the correlation between foliar area and midrib length of these 50 leaves (pvalue < 0.05) and estimated foliar area of leaves to be exposed to simulated herbivory from their midrib length and used this to determine the number of holes necessary to remove 15% of the leaf blade for the damaged leaves. Before herbivory simulation, and 60 days after the seedling transplantation, the number of fully-expanded leaves was recorded for each plant. We then simulated herbivory on 50% of the leaves of each individual, and exclusively on those located on the distal section of the stem. Young, not fully-expanded leaves were entirely removed before herbivory simulation so that ten leaves exclusively developed after the treatment could be easily identified. This young leaf removal was not conducted on control plants, which remained intact. Therefore, after the development and complete expansion of the tenth leaf after herbivory simulation, the set of ten leaves was collected to measure the herbivory-dependent defense response (i.e., EFN density based on the relationship between foliar area and EFN number). In undamaged control plants, the ten distal fully-expanded leaves were collected at the end of the experimental period. Therefore, these plants were kept without any kind of damage. Foliar area was measured using AxioVision SE64 (AxioVision Microscopy Software, 2009; ZEISS, Germany). For EFNs density measurements, we employed entire leaf blades.
2. Material and methods 2.1. Seedlings and plant material Ninety seedlings were obtained from seeds germinated in plastic seed pot trays with a vermiculite substrate that was watered regularly to maintain humidity. When seedlings exhibited two developed leaves they were transferred into 3-L plastic pots filled with hydroponic nutrient solution based on the original recipe of Hoagland and Arnon (1950) but using Fe-EDTA solution instead of iron tartrate as an iron source. To facilitate transplantation, seedlings were rinsed in deionized water, to remove all sediments on the roots, and then conditioned in pots coated with aluminized ink and containing three liters of nutrient solution. During the experimental period, pots with plants were
2.4. Nectar chemical analysis We collected secreted nectar from all EFNs of one fully-expanded leaf of three randomly selected individuals per nutritional concentration and herbivory treatment. As the amount of nectar was small and difficult to measure, we assembled the three-individual collections in a single sample. For nectar collection, we excluded all ants on every plant and subsequently washed and bagged (plastic bags) all leaves at sunset 2
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to create a wet chamber and prevent access by nectar consumers and nectar evaporation. These plastic-wet chambers were inaccessible to nectar consumers like ants present at the greenhouse. Nectar was collected during the next morning (07:00AM), approx. 12 hours after bagging leaves, by using a capillary tube. Nectar was removed from all nectaries once a time, from different leaves; subsequently we put the samples in plastic microtubes (Eppendorf, 1 mL), which were stored in a freezer at −80 °C. For nectar analysis, we used the enzymatic method to quantify the amount of glucose, fructose and sucrose present (Praxedes et al., 2006). The following enzymes were used: sucrose-phosphate synthase (sucrose), fructose 1,6-biphosphatase (fructose) and ADP-glucose pyrophosphorylase (glucose). The trials were executed using an ELISA microplate reader (Tunable Microplate Reader, VERSAmax, Sunnyvale, USA) following Praxedes et al. (2006). We did not test any statistical significance for this analysis due to the small sample size.
Table 1 Results of the nested ANOVAs with factorial experimental design testing the effects of nutritional status, herbivory (fixed factors), plant (random factor) and the interaction between nutritional status and herbivory (fixed factor) on foliar area and EFN density in Luffa cylindrica. Response
Effect
df
MS
F
p-value
Foliar area
Nutrients Herbivory Nutrients x herbivory Plant Error Nutrients Herbivory Nutrients x herbivory Plant Error
2 1 2 84 810 2 1 2 84 810
1.00 1.83 8.33 0.15 0.03 4.90 7.27 5.34 0.16 0.03
6.7 12.2 55.6 5.6
0.002 0.001 < 0.001 < 0.001
31.1 46.3 34.0 5.2
< 0.001 < 0.001 < 0.001 < 0.001
Density of EFNs
2.5. Statistical analysis To determine whether EFN density was affected by nutritional status and herbivory, we conducted two ANOVAs (i.e. for foliar area and EFN density as response variables, respectively) with a random factor (plant) nested within the two fixed-factor (herbivory and nutrients) crossed experimental design (Zar, 2010). Therefore, we included herbivory, nutrients, herbivory × nutrients (fixed factors) and plant within herbivory × nutrients (random factor) as independent variables in our model. A significant interaction indicates a differential effect of herbivory on the response variables depending on nutrient concentration. To test significant differences among nutrient concentrations under herbivory treatments for each response variable, means were compared by using post-hoc Tukey HSD. Assumptions of normality and homoscedasticity of variance were tested with ShapiroWilk and Levene tests, respectively. Then foliar area and EFN density data were log-transformed to improve normality. To determine significant leaf ontogenetic variation over time in the response of induced indirect defense of plants (i.e. between the most basal and apical leaves developed after the damage), paired t-tests were conducted for each factor and treatment (nutrient concentration and herbivory presence) to test differences in the density of EFNs between the first and the last expanded leaf collected after the experiment (i.e. between leaves 1 and 10). Mean values from all individuals for each leaf (i.e. from 1 to 10) were used (N = 15 individuals). Overall, six paired ttests were conducted (10, 50 and 100% of nutrient concentration with or without herbivory) in SPSS v22 (IBM SPSS Statistics, 2013; IBM Corp, Armonk, NY, USA). 3. Results 3.1. Effects of nutritional status and herbivory Foliar area and EFN density differed significantly among nutritional status, herbivory and plant. Moreover, the effect of herbivory on foliar area and EFN density had a significant dependence on nutrient concentration (significant nutrient × herbivory interaction; Table 1, Fig. 1). For damaged plants, leaf area significantly increased (mean ± SE: 49.07 cm−2 ± 1.47 and 57.93 cm−2 ± 1.37 for undamaged and damaged plants, respectively), whereas EFN density significantly decreased (1.59 ± 0.04 and 1.20 ± 0.04 for undamaged and damaged plants, respectively). In undamaged plants, foliar area increased in proportion with nutritional status (i.e., the responses in terms of size increased in relation to nutrient increase; Fig. 1A). EFN density in these plants tended to be similar among different nutrient concentrations, except for 50% (Fig. 1B). In damaged plants, foliar area decreased with nutrient concentration, and so was significantly larger at 10% but significantly smaller at 100%, than that of undamaged plants (Fig. 1A). EFN density under the effects of herbivory increased
Fig. 1. Variation in foliar area and EFN density in Luffa cylindrica based on differences in nutritional status and the effect of herbivory. Columns denote means while bars denote standard errors. Different letters show significant differences among treatments using a post-hoc Tukey HSD (p-value < 0.05).
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treatments of nutritional status and herbivory due to the low sample size. In damaged plants, there was an increase of about 40% and 130% of secreted sugars at 10% and 100% of nutrient concentration, respectively, in relation to undamaged plants. Sugar concentration between herbivory treatments was similar at 50% nutrient concentration. The results suggest that the combined action of resource availability and herbivory tended to result in increased sugar concentration, but our sample size was low and statistical tests were not applied, so interpretation should be made with caution. 4. Discussion This study demonstrates that EFN density related to herbivore-induced indirect defense is a damage- and resource-dependent response in L. cylindrica. It is widely known that increases in plant defense responses involve direct costs in terms of resource allocation and, consequently, possible limited availability for future defense and subsequent individual fitness (Rautio et al., 2005; Gao et al., 2008; Sun et al., 2009). Other studies have also found that indirect defenses related to predator attractiveness increase with herbivore incidence, thus being damage-dependent (Mondor and Addicott, 2003; Ness, 2003; Karban and Baldwin, 2007; Heil et al., 2009). However, the combined effect of resource availability and presence/absence of herbivory on the production of induced defense by secretory structures, such as EFNs, has received less attention (Mondor et al., 2006). Additionally, our results support the assumption that EFN density related to herbivoreinduced indirect defense is temporally variable (Heil et al., 2004; Karban, 2011; Dáttilo et al., 2015). We discuss the intensity of EFN responses based on variation in nutritional status and herbivory incidence in L. cylindrica and their ecological implications in plant-herbivore interactions. In L. cylindrica, foliar area increased with nutrient concentration in the absence of herbivory. As expected, plants with greater nutrient input showed larger foliar area, due to increases in resource availability (Knops and Reinhart, 1999; Fageria et al., 2009). Likewise, plants exhibit lower biomass under the effects of herbivory (reviewed in Strauss et al., 2002); hence, undamaged plants would not experience the negative selective pressure on foliar size exerted by herbivores. We found a trend to maintain a similar density of EFNs across the nutrient gradient in undamaged plants (i.e. the EFN number proportionally increased with the foliar area). Although increasing the number of EFNs is directly related to resource availability (Stamp, 2003; Mondor et al., 2006; Karban, 2011; Yamawo et al., 2014), its production under damage absence trends to keep constant regardless of nutrient input (Mondor et al., 2006) and it would rather decrease to avoid tradeoffs with other functions, such as foliar area development (Quintero et al., 2013 and references therein). A plausible explanation for the reported pattern is that larger leaves could potentially be more prone to attack (Brown and Lawton, 1991; Cárdenas et al., 2014) and maintenance of similar densities of nectaries would readily provide an effective defense (Mondor et al., 2006; Lortzing et al., 2016). Overall, our results show the importance of reporting EFN density as an honest measure of induced indirect defense when combined with variation in leaf area. Under simulated herbivory, foliar area was significantly smaller than in undamaged plants only for the control nutrient concentration (i.e. 100% of nutrients). Since herbivore-mediated selection tends to reduce plant organ size (Strauss and Agrawal, 1999; Strauss et al., 2002), we predicted that leaves would be smaller for all nutrient concentrations under the effects of herbivory. Likewise, foliar area from damaged plants was significantly larger in plants cultivated with 10% of nutrient concentration than in those with 50 and 100%. Overall, these findings contrast with leaf size reduction-based expectations under the effects of herbivory and resource limitation (Stamp, 2003; Karban, 2011) and seem, at first glance, to be unexpected and difficult to explain. However, mechanisms of overcompensation and their effects on plant development may help to disentangle the patterns reported
Fig. 2. Ontogenetic variation over time (i.e. between the most basal and the apical leaves developed after the damage) in EFN density at the end of the experiment for undamaged (control) and damaged (herbivory simulation) plants in each nutritional status in Luffa cylindrica. Columns denote means while bars denote standard errors. * p-value < 0.05.
with nutrient concentration and tended to exceed the EFN density of undamaged plants under nutrient-control conditions (Fig. 1B). 3.2. Ontogenetic variation Ontogenetic significant variation over time (i.e. between the most basal and the apical leaves developed after the damage) in EFN density was only detected at 10% of nutrient concentration (Fig. 2). In undamaged plants, the average values were significantly 29% higher in apical leaves (mean ± SD: 1.57 ± 0.40 vs 2.21 ± 0.69, paired t-test1, 14 = −2.80, p-value = 0.014; Fig. 2). Likewise, there was a significant increase of 92% in EFN density over time (i.e. in the apical leaves) in plants with simulated herbivory (mean ± SD: 0.45 ± 0.21 vs 0.86 ± 0.34, paired t-test1, 14 = −4.78, p-value < 0.001; Fig. 2). A marginal increase of 36% in EFN density in the apical leaves was also detected in undamaged plants at 100% of nutrient concentration (mean ± SD: 1.28 ± 0.68 vs 1.75 ± 0.70, paired t-test1, 14 = −2.02, p-value < 0.063; Fig. 2). 3.3. Nectar composition On average (mean ± SD), sugar concentration in extrafloral nectar comprised 39.3 ± 13.1 mg/ml (22.6 ± 6.8, 8.9 ± 3.9 and 7.8 ± 3.3 mg/ml for sucrose, fructose and glucose, respectively; Fig. 3). However, we did not test for statistical significance among
Fig. 3. Sugar concentrations in nectar of Luffa cylindrica in response to differences in nutritional status and herbivory treatment. 4
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sugar concentrations in plants under 100% of nutrient concentration and the effects of herbivory (Fig. 3). Since nectar secretion is costly in terms of resources, and is induced by herbivores, this result agrees with the damage- and resource-dependent response and optimal defense theory (McKey, 1974; Stamp, 2003; Mondor et al., 2006; Xu and Chen, 2015). It is also noteworthy that damaged plants with 10% of nutrients also possessed high sugar concentrations in nectar. Overcompensatory responses and higher risks of fitness reduction under this low nutritional status could help to explain this result (Agrawal, 2000). Increases in nectar-sugar secretion are related to higher herbivore incidence (Heil and McKey, 2003), in that improved nectar quality increases subsequent predator attraction (e.g. ants: Pulice and Packer, 2008; Byk and Del Claro, 2011; Dáttilo et al., 2015). In this way, plants with nutritional deficiencies and suffering mechanical damage may make a high investment in nectar and intensify ant attraction to lengthen their patrolling time to avoid photosynthetic tissue loss. The degree to which resource availability and herbivory damage affect nectar quality and the subsequent predator-mediated defense in Luffa, warrants further investigation. In undamaged plants, Xu and Chen (2015) showed that the amount of EFN secretion was positively correlated to average photosynthesis rate. In conclusion, our results contribute to understanding the factors that modulate indirect defense in plants, such as nutrient availability and herbivory, by means of plastic responses of EFNs in Luffa cylindrica. We show experimental evidence that plant defense mediated by changes in EFN response in this species is damage- and resource-dependent. Furthermore, we demonstrate that this pattern shows ontogenetic variation over time under different nutritional status and presence/absence of herbivory, an under-appreciated topic in multitrophic plant-herbivore-predator interactions. We suggest that further research into the effects of interactions between different levels of mechanical damage and resource availability, and subsequent specific analyses of nectar quantity and quality, is warranted in order to generate solid predictions about the effects of multi-species interactions on plant defense responses and the resulting plant fitness.
here and to better interpret our results. As reported for other monocarpic plants, an overcompensatory response derived from a combined effect of herbivory and resource limitation in L. cylindrica may be plausible given the opportunity for a bet-hedging strategy within a single flowering season and reproductive bout without compromising future growth, reproduction and survival (Paige and Whitham, 1987; Vail, 1992; Rautio et al., 2005). Therefore, we suggest that under the effects of herbivory, more stressed plants under nutrient-poor conditions may show positive responses, such as larger foliar area than plants with high nutritional status, in order to increase photosynthetic area or even photosynthetic rates (see Schwachtje and Baldwin, 2008). Alternatively (or jointly), divergent development of traits related to plant growth and defense under stressful conditions could lead to negative phenotypic correlations (i.e. trade-offs) and potentially explain the decreased foliar area in damaged plants with increasing nutrient concentrations (Agrawal, 2011). In this regard, leaf damage under nutrient-poor conditions may induce a low production of EFNs, which would concurrently allow resource reallocation to leaf expansion (Yamawo et al., 2015; Yamawo, 2017). Conversely, a higher nutrient acquisition under the effects of herbivory may provide an increased and less-limited indirect defense response, but at the expenses of a reduced foliar area. This potential trade-off between multiple traits related to plant growth and defense opens the possibility that, in L. cylindrica, the induction of indirect defense traits such as EFNs is costlier than investment to leaf development and photosynthetic tissue. In agreement to this, we demonstrated an increase in EFN density under the effects of herbivory with increasing nutrient concentration. This also supports the assumptions that induced plant defense responses depend on herbivory and resource availability (Stamp, 2003; Mondor et al., 2006; Yamawo et al., 2012, 2014). Although this result is predictable, we also identified a reduction in EFN density in relation to undamaged plants, especially under nutrient-poor conditions. This result is difficult to explain based on the damage-dependent theory for EFN production related to herbivore-induced indirect response (Stamp, 2003; Mondor et al., 2006; Lortzing et al., 2016). In this regard, the decrease in the density of EFNs found on damaged plants in relation to undamaged ones treated with nutrient concentrations of 10% and 50% seems to be a feasible consequence of the overcompensatory response biased towards larger leaf area and subsequent reduction in defense investment due to resource limitation and functional tradeoffs in L. cylindrica. However, we suggest that future research should analyze different types of simulated damage (e.g. scratching, punching and cutting off full leaves; Xu et al., 2014) and levels of leaf area removal (e.g. 0, 25, 50 and 75%; Gao et al., 2008, 2014) along with a nutrient gradient to assess the effects of any interaction between type and level of leaf damage and resource availability on EFN density. An important finding in our study was the ontogenic variation in EFN density in response to herbivore-induced indirect defense (Fig. 2). Plants cultivated under 10% nutrient concentration showed a temporal increase in EFN density after simulated herbivory. This pattern seems to be expected since younger leaves are more vulnerable to attack and, consequently, plants optimize resource allocation to produce a greater number of EFNs on apical leaves (Heil et al., 2004; Dáttilo et al., 2015). This result contrasts with response peaks in EFN activity over time, as has been reported for other species (e.g. Qualea multiflora, Calixto et al., 2015). At 10% nutrient concentration, the response of an increased density of EFNs is also in agreement with an overcompensatory response, as pointed out above for this nutritional status under the effects of herbivory (Agrawal, 2000). For undamaged plants, we detected a stabilizing trend in EFN density over time under 10% of nutrient concentration, showing an initial reduction followed by a significant increase. As pointed above, this pattern may be closely related to a direct relationship between the number of EFNs and foliar area and the higher probabilities of herbivore incidence on larger leaves, thus maintaining the EFN density. With regard to nectar content, we detected a tendency for increased
Author's contribution EASP conceived the main idea. EASP and PFSL conceived and planned the experiments. PFSL carried out the experiments, measurements and data collection. ALT and PFSL analyzed the data. All authors discussed the results, contributed to the final manuscript and have approved the final version of the article. Funding ALT receives a post-doctoral scholarship form Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and EASP receives a research grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (306790-2015-7). PFSL received a Master scholarship from Fundação de Amparo à Pesquisa do Estado de Minas Gerais. Acknowledgements We thank two anonymous reviewers for the comments provided to the text and to Dr. FAO Silveira for his help during experiment implementation. We also thank the staff of the Laboratory of Plant Nutrition and Metabolism, Department of Plant Biology, Federal University of Viçosa (Viçosa, MG, Brazil) for helping with nectar analysis. References Agarwal, V.M., Rastogi, N., 2010. Ants as dominant insect visitors of the extrafloral nectaries of sponge gourd plant, Luffa cylindrica (L.) (Cucurbitaceae). Asian Myrmecol 3, 45–54.
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Herbivory-induced overcompensation and resource-dependent production of extrafloral nectaries in Luffa cylindrica (Cucurbitaceae)
T
Poliana Fernandes Souza Limaa, Alberto L. Teixidoa, Elder Antônio Sousa Paivaa,∗ a
Departamento de Botânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 30161-970, Belo Horizonte, Minas Gerais, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Damage-dependent response EFN density Extrafloral nectary Leaf area Plant nutritional status Resource-dependent response
Extrafloral nectaries (EFNs) are nectar secretory structures involved in the indirect defense of plants. In the sponge gourd (Luffa cylindrica), EFNs commonly occur on the lower surface of leaf blades and stipules and remain functional until leaf senescence. To test the hypothesis that the development of EFNs is influenced by herbivore damage and resource availability, we grew Luffa cylindrica under different concentrations of Hoagland's nutrient solution (nutrient-poor conditions: 10%, 50%; and control condition: 100%) and two herbivory treatments (damaged and undamaged leaves). We collected ten leaves from treated plants to quantify leaf area and EFN density. Overall, leaf area increased and EFN decreased in damaged plants, but this significantly depended on nutritional status. In undamaged plants, EFN density tended to remain constant, whereas foliar area increased with nutrient input. Under herbivory, foliar area increased at 10% but decreased at 50 and 100% of nutrients in relation to undamaged plants, whereas EFN density tended to increase with nutrient availability to exceed undamaged plants under control concentrations. Plants under nutrient-poor conditions subjected to herbivory exhibited an increased foliar area, characterizing a compensatory mechanism. Our results suggest that herbivore-induced indirect defense is a damage- and resource-dependent response in Luffa cylindrica. These findings contribute to understanding the factors that modulate indirect defenses and plant-herbivore-environment interactions.
1. Introduction Plants have to deal with a diverse assemblage of herbivores, which can consume significant amounts of biomass and reduce plant reproductive success (Crawley, 1983; Coley and Barone, 1996; Strauss et al., 2002; Turcotte et al., 2014). Consequently, plants have developed a diversity of structures and evolutionary strategies to provide protection against herbivory. For example, some mechanisms provide indirect defense by attracting natural predators of herbivores (Agrawal, 1998; Karban and Baldwin, 2007; Yamawo, 2017). However, resource allocation to defense may compromise plant development and reproduction (Strauss and Agrawal, 1999; Schiestl et al., 2014). Thus, induced defenses stimulated at the moment of damage have been favored by resource-mediated natural selection (Zangerl and Rutledge, 1996; Ness, 2003; Meldau et al., 2012; Lortzing et al., 2016), which entail a resource cost only when strictly necessary and thus allow increased allocation of resources to other functions. Herbivore-induced defense strategies of plants are generally resource-dependent (Stamp, 2003; Mondor et al., 2006; Karban, 2011; Yamawo, 2017). Following this assumption, resource allocation
∗
patterns for induced defenses mostly follow optimal defense theory, which suggests that plant organs and structures with a high energetic value and a subsequent higher herbivore incidence show higher investment in defense (McKey, 1974; Mondor et al., 2006; Xu and Chen, 2015). Therefore, one could experimentally evaluate whether both resource availability and mechanical damage significantly affect the induction of defenses. Evidence in this sense would potentially explain reduced defense responses in the absence of damage and in resourcepoor conditions. In some cases, the development and reproduction of plants damaged by herbivores can be even greater than that of undamaged plants. Such a positive effect of herbivory on plant performance is referred to as a mechanism of overcompensation. Herbivore-mediated selection for overcompensatory responses favors increased fitness of damaged plants and plant traits that increase the probability of attack (Paige and Whitham, 1987; Vail, 1992; Agrawal, 2000; Poveda et al., 2010). The benefits derived from overcompensation are associated with increased lateral branching, stimulated growth and subsequent improved plant reproduction (Agrawal, 2000; Rautio et al., 2005; Sun et al., 2009; Olejniczak, 2011). Interestingly, plant compensatory responses to
Corresponding author. E-mail address: [email protected] (E.A. Sousa Paiva).
https://doi.org/10.1016/j.actao.2018.10.001 Received 23 March 2018; Received in revised form 15 August 2018; Accepted 9 October 2018 1146-609X/ © 2018 Elsevier Masson SAS. All rights reserved.
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randomly arranged in a greenhouse where they were maintained under a 12/12 hours light/dark cycle with a light intensity of 400 μmol/m−2/ s−1.
herbivory may also be affected by resource availability; however, although it is still unclear whether higher resource levels increase, or not, tolerance of herbivory, it appears to be largely dependent on the most limiting resources (see Wise and Abrahamson, 2007). Some of herbivore-induced indirect defense strategies comprise a diversity of secretory structures that excrete chemical compounds that promote predator-mediated protection against herbivory (Stamp, 2003; Heil, 2008; Nahas et al., 2012; Lortzing et al., 2016). A broad taxonomic array of species is known to produce extrafloral nectaries (hereafter, EFNs; Heil et al., 2009; Weber and Keeler, 2013; Weber et al., 2015). EFNs are structures involved in nectar secretion, a valuable energetic resource for arthropod species that reduce the presence of herbivores and other plant enemies (Heil, 2011). Therefore, EFNs are not directly linked to pollinator attraction, but rather they are commonly responsible for indirect biotic defense of plants by attracting other species such as ants and wasps, which by means of their presence and activity promote plant protection (Schmid, 1988; Byk and Del Claro, 2011; Gonçalves-Souza et al., 2016). In this regard, EFNs are key elements for increased biomass, leaf production, reproduction and fitness of plants (reviewed in Rosumek et al., 2009). EFNs are also herbivore-induced structures largely dependent on resource availability (Mondor et al., 2006; Karban, 2011; Yamawo et al., 2012, 2014), so EFN production can readily decrease along plant ontogeny due to tradeoffs with other functions or changes in its inducibility, but generally increasing under damage events (Quintero et al., 2013 and references therein). Therefore, changes in production of EFNs are essential to understand the induction of indirect defenses related to herbivore damage and resource dependence. The gourd family, Cucurbitaceae, is especially rich in EFN-bearing species, including the annual and monoecious sponge gourd (Luffa cylindrica), which possesses disc-shaped EFNs on its leaf surface, stipules, bracts, bracteoles and fruits (Agarwal and Rastogi, 2010; Nesom, 2015). These EFNs are conspicuous, numerous (reaching 80 by leaf blade) and produce nectar as droplets, mainly during the stages of leaf expansion and in young expanded leaves. Associated with this species is a highly diverse set of insect herbivores and their natural predators, in particular, ants are the most frequent group (Agarwal and Rastogi, 2010). The number of EFNs in L. cylindrica is positively correlated with predator ant patrolling time, representing a relevant indirect defense strategy (Agarwal and Rastogi, 2010). Here we aim to understand the relationship between nutritional status and defense investment by examining foliar area and EFN density of L. cylindrica cultivated under different nutrient concentrations and mechanical damages. We exclusively focused on EFN density as it is a true putative measure of defense readily comparable with previous studies showing herbivory- and resource-dependent EFN production (e.g. Mondor et al., 2006; Lortzing et al., 2016). We specifically tested if: (1) leaf area and EFN density vary with the nutritional status and simulated herbivory; and (2) EFNs density varies along plant ontogeny between the most basal and apical leaves developed after the simulated herbivory.
2.2. Nutrient solution To evaluate whether EFN density is damage- and resource-dependent, three nutrient solutions of different concentrations (10, 50 and 100% strength of Hoagland nutrient solution) were used, on 30 randomly selected seedlings. The 100% concentration was used as nutrient control group, since the plants received normal and appropriate levels of mineral nutrients, whereas the concentrations of 10 and 50% of nutrients were used as experimental treatments (reduction of resources). The volume of liquid in the pots was filled daily with deionized water to replace loss by evaporation, and the solution was renewed weekly to maintain the experimental nutrient concentrations, until the end of experimental period (80 days). 2.3. Herbivory To determine whether EFN density can be induced by mechanical damage, we simulated herbivory on 15 plants under each nutrient treatment (i.e. 10, 50 and 100%) 60 days after seedling transplantation, whereas 15 plants in each nutrient condition were unmanipulated and left as controls. All plants, regardless of treatment, were in a non-reproductive stage. Simulation of herbivory consisted of removal of 15% of the foliar area by means of a single-hole punch (Staples 10573-CC, Staples Inc, SP, Brazil). Although the amount of foliar area to be removed to simulate herbivory is somewhat variable in the literature, 15% fits the mean damage to plants in natural communities (Strauss and Agrawal, 1999) and was sufficient to test our hypotheses (see Results). Foliar area was previously assessed for 50 randomly chosen leaves from adult plants by using AxioVision SE64 (AxioVision Microscopy Software, 2009; ZEISS, Germany). We subsequently estimated the correlation between foliar area and midrib length of these 50 leaves (pvalue < 0.05) and estimated foliar area of leaves to be exposed to simulated herbivory from their midrib length and used this to determine the number of holes necessary to remove 15% of the leaf blade for the damaged leaves. Before herbivory simulation, and 60 days after the seedling transplantation, the number of fully-expanded leaves was recorded for each plant. We then simulated herbivory on 50% of the leaves of each individual, and exclusively on those located on the distal section of the stem. Young, not fully-expanded leaves were entirely removed before herbivory simulation so that ten leaves exclusively developed after the treatment could be easily identified. This young leaf removal was not conducted on control plants, which remained intact. Therefore, after the development and complete expansion of the tenth leaf after herbivory simulation, the set of ten leaves was collected to measure the herbivory-dependent defense response (i.e., EFN density based on the relationship between foliar area and EFN number). In undamaged control plants, the ten distal fully-expanded leaves were collected at the end of the experimental period. Therefore, these plants were kept without any kind of damage. Foliar area was measured using AxioVision SE64 (AxioVision Microscopy Software, 2009; ZEISS, Germany). For EFNs density measurements, we employed entire leaf blades.
2. Material and methods 2.1. Seedlings and plant material Ninety seedlings were obtained from seeds germinated in plastic seed pot trays with a vermiculite substrate that was watered regularly to maintain humidity. When seedlings exhibited two developed leaves they were transferred into 3-L plastic pots filled with hydroponic nutrient solution based on the original recipe of Hoagland and Arnon (1950) but using Fe-EDTA solution instead of iron tartrate as an iron source. To facilitate transplantation, seedlings were rinsed in deionized water, to remove all sediments on the roots, and then conditioned in pots coated with aluminized ink and containing three liters of nutrient solution. During the experimental period, pots with plants were
2.4. Nectar chemical analysis We collected secreted nectar from all EFNs of one fully-expanded leaf of three randomly selected individuals per nutritional concentration and herbivory treatment. As the amount of nectar was small and difficult to measure, we assembled the three-individual collections in a single sample. For nectar collection, we excluded all ants on every plant and subsequently washed and bagged (plastic bags) all leaves at sunset 2
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to create a wet chamber and prevent access by nectar consumers and nectar evaporation. These plastic-wet chambers were inaccessible to nectar consumers like ants present at the greenhouse. Nectar was collected during the next morning (07:00AM), approx. 12 hours after bagging leaves, by using a capillary tube. Nectar was removed from all nectaries once a time, from different leaves; subsequently we put the samples in plastic microtubes (Eppendorf, 1 mL), which were stored in a freezer at −80 °C. For nectar analysis, we used the enzymatic method to quantify the amount of glucose, fructose and sucrose present (Praxedes et al., 2006). The following enzymes were used: sucrose-phosphate synthase (sucrose), fructose 1,6-biphosphatase (fructose) and ADP-glucose pyrophosphorylase (glucose). The trials were executed using an ELISA microplate reader (Tunable Microplate Reader, VERSAmax, Sunnyvale, USA) following Praxedes et al. (2006). We did not test any statistical significance for this analysis due to the small sample size.
Table 1 Results of the nested ANOVAs with factorial experimental design testing the effects of nutritional status, herbivory (fixed factors), plant (random factor) and the interaction between nutritional status and herbivory (fixed factor) on foliar area and EFN density in Luffa cylindrica. Response
Effect
df
MS
F
p-value
Foliar area
Nutrients Herbivory Nutrients x herbivory Plant Error Nutrients Herbivory Nutrients x herbivory Plant Error
2 1 2 84 810 2 1 2 84 810
1.00 1.83 8.33 0.15 0.03 4.90 7.27 5.34 0.16 0.03
6.7 12.2 55.6 5.6
0.002 0.001 < 0.001 < 0.001
31.1 46.3 34.0 5.2
< 0.001 < 0.001 < 0.001 < 0.001
Density of EFNs
2.5. Statistical analysis To determine whether EFN density was affected by nutritional status and herbivory, we conducted two ANOVAs (i.e. for foliar area and EFN density as response variables, respectively) with a random factor (plant) nested within the two fixed-factor (herbivory and nutrients) crossed experimental design (Zar, 2010). Therefore, we included herbivory, nutrients, herbivory × nutrients (fixed factors) and plant within herbivory × nutrients (random factor) as independent variables in our model. A significant interaction indicates a differential effect of herbivory on the response variables depending on nutrient concentration. To test significant differences among nutrient concentrations under herbivory treatments for each response variable, means were compared by using post-hoc Tukey HSD. Assumptions of normality and homoscedasticity of variance were tested with ShapiroWilk and Levene tests, respectively. Then foliar area and EFN density data were log-transformed to improve normality. To determine significant leaf ontogenetic variation over time in the response of induced indirect defense of plants (i.e. between the most basal and apical leaves developed after the damage), paired t-tests were conducted for each factor and treatment (nutrient concentration and herbivory presence) to test differences in the density of EFNs between the first and the last expanded leaf collected after the experiment (i.e. between leaves 1 and 10). Mean values from all individuals for each leaf (i.e. from 1 to 10) were used (N = 15 individuals). Overall, six paired ttests were conducted (10, 50 and 100% of nutrient concentration with or without herbivory) in SPSS v22 (IBM SPSS Statistics, 2013; IBM Corp, Armonk, NY, USA). 3. Results 3.1. Effects of nutritional status and herbivory Foliar area and EFN density differed significantly among nutritional status, herbivory and plant. Moreover, the effect of herbivory on foliar area and EFN density had a significant dependence on nutrient concentration (significant nutrient × herbivory interaction; Table 1, Fig. 1). For damaged plants, leaf area significantly increased (mean ± SE: 49.07 cm−2 ± 1.47 and 57.93 cm−2 ± 1.37 for undamaged and damaged plants, respectively), whereas EFN density significantly decreased (1.59 ± 0.04 and 1.20 ± 0.04 for undamaged and damaged plants, respectively). In undamaged plants, foliar area increased in proportion with nutritional status (i.e., the responses in terms of size increased in relation to nutrient increase; Fig. 1A). EFN density in these plants tended to be similar among different nutrient concentrations, except for 50% (Fig. 1B). In damaged plants, foliar area decreased with nutrient concentration, and so was significantly larger at 10% but significantly smaller at 100%, than that of undamaged plants (Fig. 1A). EFN density under the effects of herbivory increased
Fig. 1. Variation in foliar area and EFN density in Luffa cylindrica based on differences in nutritional status and the effect of herbivory. Columns denote means while bars denote standard errors. Different letters show significant differences among treatments using a post-hoc Tukey HSD (p-value < 0.05).
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treatments of nutritional status and herbivory due to the low sample size. In damaged plants, there was an increase of about 40% and 130% of secreted sugars at 10% and 100% of nutrient concentration, respectively, in relation to undamaged plants. Sugar concentration between herbivory treatments was similar at 50% nutrient concentration. The results suggest that the combined action of resource availability and herbivory tended to result in increased sugar concentration, but our sample size was low and statistical tests were not applied, so interpretation should be made with caution. 4. Discussion This study demonstrates that EFN density related to herbivore-induced indirect defense is a damage- and resource-dependent response in L. cylindrica. It is widely known that increases in plant defense responses involve direct costs in terms of resource allocation and, consequently, possible limited availability for future defense and subsequent individual fitness (Rautio et al., 2005; Gao et al., 2008; Sun et al., 2009). Other studies have also found that indirect defenses related to predator attractiveness increase with herbivore incidence, thus being damage-dependent (Mondor and Addicott, 2003; Ness, 2003; Karban and Baldwin, 2007; Heil et al., 2009). However, the combined effect of resource availability and presence/absence of herbivory on the production of induced defense by secretory structures, such as EFNs, has received less attention (Mondor et al., 2006). Additionally, our results support the assumption that EFN density related to herbivoreinduced indirect defense is temporally variable (Heil et al., 2004; Karban, 2011; Dáttilo et al., 2015). We discuss the intensity of EFN responses based on variation in nutritional status and herbivory incidence in L. cylindrica and their ecological implications in plant-herbivore interactions. In L. cylindrica, foliar area increased with nutrient concentration in the absence of herbivory. As expected, plants with greater nutrient input showed larger foliar area, due to increases in resource availability (Knops and Reinhart, 1999; Fageria et al., 2009). Likewise, plants exhibit lower biomass under the effects of herbivory (reviewed in Strauss et al., 2002); hence, undamaged plants would not experience the negative selective pressure on foliar size exerted by herbivores. We found a trend to maintain a similar density of EFNs across the nutrient gradient in undamaged plants (i.e. the EFN number proportionally increased with the foliar area). Although increasing the number of EFNs is directly related to resource availability (Stamp, 2003; Mondor et al., 2006; Karban, 2011; Yamawo et al., 2014), its production under damage absence trends to keep constant regardless of nutrient input (Mondor et al., 2006) and it would rather decrease to avoid tradeoffs with other functions, such as foliar area development (Quintero et al., 2013 and references therein). A plausible explanation for the reported pattern is that larger leaves could potentially be more prone to attack (Brown and Lawton, 1991; Cárdenas et al., 2014) and maintenance of similar densities of nectaries would readily provide an effective defense (Mondor et al., 2006; Lortzing et al., 2016). Overall, our results show the importance of reporting EFN density as an honest measure of induced indirect defense when combined with variation in leaf area. Under simulated herbivory, foliar area was significantly smaller than in undamaged plants only for the control nutrient concentration (i.e. 100% of nutrients). Since herbivore-mediated selection tends to reduce plant organ size (Strauss and Agrawal, 1999; Strauss et al., 2002), we predicted that leaves would be smaller for all nutrient concentrations under the effects of herbivory. Likewise, foliar area from damaged plants was significantly larger in plants cultivated with 10% of nutrient concentration than in those with 50 and 100%. Overall, these findings contrast with leaf size reduction-based expectations under the effects of herbivory and resource limitation (Stamp, 2003; Karban, 2011) and seem, at first glance, to be unexpected and difficult to explain. However, mechanisms of overcompensation and their effects on plant development may help to disentangle the patterns reported
Fig. 2. Ontogenetic variation over time (i.e. between the most basal and the apical leaves developed after the damage) in EFN density at the end of the experiment for undamaged (control) and damaged (herbivory simulation) plants in each nutritional status in Luffa cylindrica. Columns denote means while bars denote standard errors. * p-value < 0.05.
with nutrient concentration and tended to exceed the EFN density of undamaged plants under nutrient-control conditions (Fig. 1B). 3.2. Ontogenetic variation Ontogenetic significant variation over time (i.e. between the most basal and the apical leaves developed after the damage) in EFN density was only detected at 10% of nutrient concentration (Fig. 2). In undamaged plants, the average values were significantly 29% higher in apical leaves (mean ± SD: 1.57 ± 0.40 vs 2.21 ± 0.69, paired t-test1, 14 = −2.80, p-value = 0.014; Fig. 2). Likewise, there was a significant increase of 92% in EFN density over time (i.e. in the apical leaves) in plants with simulated herbivory (mean ± SD: 0.45 ± 0.21 vs 0.86 ± 0.34, paired t-test1, 14 = −4.78, p-value < 0.001; Fig. 2). A marginal increase of 36% in EFN density in the apical leaves was also detected in undamaged plants at 100% of nutrient concentration (mean ± SD: 1.28 ± 0.68 vs 1.75 ± 0.70, paired t-test1, 14 = −2.02, p-value < 0.063; Fig. 2). 3.3. Nectar composition On average (mean ± SD), sugar concentration in extrafloral nectar comprised 39.3 ± 13.1 mg/ml (22.6 ± 6.8, 8.9 ± 3.9 and 7.8 ± 3.3 mg/ml for sucrose, fructose and glucose, respectively; Fig. 3). However, we did not test for statistical significance among
Fig. 3. Sugar concentrations in nectar of Luffa cylindrica in response to differences in nutritional status and herbivory treatment. 4
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sugar concentrations in plants under 100% of nutrient concentration and the effects of herbivory (Fig. 3). Since nectar secretion is costly in terms of resources, and is induced by herbivores, this result agrees with the damage- and resource-dependent response and optimal defense theory (McKey, 1974; Stamp, 2003; Mondor et al., 2006; Xu and Chen, 2015). It is also noteworthy that damaged plants with 10% of nutrients also possessed high sugar concentrations in nectar. Overcompensatory responses and higher risks of fitness reduction under this low nutritional status could help to explain this result (Agrawal, 2000). Increases in nectar-sugar secretion are related to higher herbivore incidence (Heil and McKey, 2003), in that improved nectar quality increases subsequent predator attraction (e.g. ants: Pulice and Packer, 2008; Byk and Del Claro, 2011; Dáttilo et al., 2015). In this way, plants with nutritional deficiencies and suffering mechanical damage may make a high investment in nectar and intensify ant attraction to lengthen their patrolling time to avoid photosynthetic tissue loss. The degree to which resource availability and herbivory damage affect nectar quality and the subsequent predator-mediated defense in Luffa, warrants further investigation. In undamaged plants, Xu and Chen (2015) showed that the amount of EFN secretion was positively correlated to average photosynthesis rate. In conclusion, our results contribute to understanding the factors that modulate indirect defense in plants, such as nutrient availability and herbivory, by means of plastic responses of EFNs in Luffa cylindrica. We show experimental evidence that plant defense mediated by changes in EFN response in this species is damage- and resource-dependent. Furthermore, we demonstrate that this pattern shows ontogenetic variation over time under different nutritional status and presence/absence of herbivory, an under-appreciated topic in multitrophic plant-herbivore-predator interactions. We suggest that further research into the effects of interactions between different levels of mechanical damage and resource availability, and subsequent specific analyses of nectar quantity and quality, is warranted in order to generate solid predictions about the effects of multi-species interactions on plant defense responses and the resulting plant fitness.
here and to better interpret our results. As reported for other monocarpic plants, an overcompensatory response derived from a combined effect of herbivory and resource limitation in L. cylindrica may be plausible given the opportunity for a bet-hedging strategy within a single flowering season and reproductive bout without compromising future growth, reproduction and survival (Paige and Whitham, 1987; Vail, 1992; Rautio et al., 2005). Therefore, we suggest that under the effects of herbivory, more stressed plants under nutrient-poor conditions may show positive responses, such as larger foliar area than plants with high nutritional status, in order to increase photosynthetic area or even photosynthetic rates (see Schwachtje and Baldwin, 2008). Alternatively (or jointly), divergent development of traits related to plant growth and defense under stressful conditions could lead to negative phenotypic correlations (i.e. trade-offs) and potentially explain the decreased foliar area in damaged plants with increasing nutrient concentrations (Agrawal, 2011). In this regard, leaf damage under nutrient-poor conditions may induce a low production of EFNs, which would concurrently allow resource reallocation to leaf expansion (Yamawo et al., 2015; Yamawo, 2017). Conversely, a higher nutrient acquisition under the effects of herbivory may provide an increased and less-limited indirect defense response, but at the expenses of a reduced foliar area. This potential trade-off between multiple traits related to plant growth and defense opens the possibility that, in L. cylindrica, the induction of indirect defense traits such as EFNs is costlier than investment to leaf development and photosynthetic tissue. In agreement to this, we demonstrated an increase in EFN density under the effects of herbivory with increasing nutrient concentration. This also supports the assumptions that induced plant defense responses depend on herbivory and resource availability (Stamp, 2003; Mondor et al., 2006; Yamawo et al., 2012, 2014). Although this result is predictable, we also identified a reduction in EFN density in relation to undamaged plants, especially under nutrient-poor conditions. This result is difficult to explain based on the damage-dependent theory for EFN production related to herbivore-induced indirect response (Stamp, 2003; Mondor et al., 2006; Lortzing et al., 2016). In this regard, the decrease in the density of EFNs found on damaged plants in relation to undamaged ones treated with nutrient concentrations of 10% and 50% seems to be a feasible consequence of the overcompensatory response biased towards larger leaf area and subsequent reduction in defense investment due to resource limitation and functional tradeoffs in L. cylindrica. However, we suggest that future research should analyze different types of simulated damage (e.g. scratching, punching and cutting off full leaves; Xu et al., 2014) and levels of leaf area removal (e.g. 0, 25, 50 and 75%; Gao et al., 2008, 2014) along with a nutrient gradient to assess the effects of any interaction between type and level of leaf damage and resource availability on EFN density. An important finding in our study was the ontogenic variation in EFN density in response to herbivore-induced indirect defense (Fig. 2). Plants cultivated under 10% nutrient concentration showed a temporal increase in EFN density after simulated herbivory. This pattern seems to be expected since younger leaves are more vulnerable to attack and, consequently, plants optimize resource allocation to produce a greater number of EFNs on apical leaves (Heil et al., 2004; Dáttilo et al., 2015). This result contrasts with response peaks in EFN activity over time, as has been reported for other species (e.g. Qualea multiflora, Calixto et al., 2015). At 10% nutrient concentration, the response of an increased density of EFNs is also in agreement with an overcompensatory response, as pointed out above for this nutritional status under the effects of herbivory (Agrawal, 2000). For undamaged plants, we detected a stabilizing trend in EFN density over time under 10% of nutrient concentration, showing an initial reduction followed by a significant increase. As pointed above, this pattern may be closely related to a direct relationship between the number of EFNs and foliar area and the higher probabilities of herbivore incidence on larger leaves, thus maintaining the EFN density. With regard to nectar content, we detected a tendency for increased
Author's contribution EASP conceived the main idea. EASP and PFSL conceived and planned the experiments. PFSL carried out the experiments, measurements and data collection. ALT and PFSL analyzed the data. All authors discussed the results, contributed to the final manuscript and have approved the final version of the article. Funding ALT receives a post-doctoral scholarship form Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and EASP receives a research grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (306790-2015-7). PFSL received a Master scholarship from Fundação de Amparo à Pesquisa do Estado de Minas Gerais. Acknowledgements We thank two anonymous reviewers for the comments provided to the text and to Dr. FAO Silveira for his help during experiment implementation. We also thank the staff of the Laboratory of Plant Nutrition and Metabolism, Department of Plant Biology, Federal University of Viçosa (Viçosa, MG, Brazil) for helping with nectar analysis. References Agarwal, V.M., Rastogi, N., 2010. Ants as dominant insect visitors of the extrafloral nectaries of sponge gourd plant, Luffa cylindrica (L.) (Cucurbitaceae). Asian Myrmecol 3, 45–54.
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