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Contents lists available at ScienceDirect

Ecological Indicators

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erbivory-induced stress: Leaf developmental instability is causedy herbivore damage in early stages of leaf development

stevão Alves-Silva ∗, Kleber Del-Claronstitute of Biology, Federal University of Uberlândia, Ceará Str. 2D Building, Umuarama Campus, 38400-902 Uberlândia, Minas Gerais, Brazil

r t i c l e i n f o

rticle history:eceived 8 April 2015eceived in revised form8 September 2015ccepted 21 September 2015

eywords:anisteriopsiseteropteryserbivory-induced stresslant stress hypothesisseudophilothrips

a b s t r a c t

Herbivory is a major source of plant stress and its effects can be severe, decreasing plant fitness, or subtle,affecting the development of leaves by influencing the normal pattern of growth and expansion of leafblades. Fluctuating asymmetry (FA) analysis is recognized as a measure of plant stress, and can be usedto evaluate subtle effects of herbivory on the imperfect growth of bilaterally symmetrical traits, such asleaves. One general issue is that authors usually consider FA as an indicator of stress, which can attractherbivores (plant stress hypothesis), and studies showing that herbivores themselves affect leaf sym-metry (herbivory-induced stress hypothesis) are scarce, with mixed results. Here, we investigated therelationship between herbivory by thrips and leaf FA in Banisteriopsis malifolia and Heteropterys escalloni-folia (Malpighiaceae). Pseudophilothrips obscuricornis is a free-living, non-pest, sucking species that feedsmainly on leaf buds. We hypothesized that herbivory by thrips in the early stages of leaf developmentwould provoke increased FA levels in mature leaves. The results showed that thrips herbivory rate waslow, affecting barely more than 1% of the leaf blade. Nonetheless, thrips-attacked leaves of B. malifolia andH. escallonifolia presented increases of 15 and 27% in leaf asymmetry, respectively, compared to uninjured

leaves, corroborating the herbivory-induced stress hypothesis. Since herbivory by thrips in leaf buds wasrelated to significant increases in the stress of mature leaves, we assume that under these circumstances,FA can be used as a biomarker for plant stress following herbivory damage. To be useful as a biomarkerof stress, FA in plants must be investigated with caution, taking into account the natural history of theherbivore species and timing of leaf damage.

© 2015 Elsevier Ltd. All rights reserved.

. Introduction

Plants are distributed along a gradient of stressful conditionsCuevas-Reyes et al., 2011a) and may suffer from physiological ten-ions that might eventually cause small deviations from perfectymmetry in otherwise bilaterally symmetrical traits (Cornelissennd Stiling, 2010; Klingenberg, 2003). One common measure ofevelopmental instability is fluctuating asymmetry (FA) analysis,

component of character size variation widely used to estimate aopulation’s response in relation to stressful conditions (Palmer,996; Palmer and Strobeck, 1986). For instance, FA in plants isssociated with a wide range of environmental variables, suchs pollution (Zvereva et al., 1997a), soil salinity (Cornelissen and

Please cite this article in press as: Alves-Silva, E., Del-Claro, K., Herbiherbivore damage in early stages of leaf development. Ecol. Indicat. (2

tiling, 2010), shade conditions (Puerta-Pinero et al., 2008) andlimate (Cowart and Graham, 1999), among others. In these cir-umstances, leaves present increased levels of difference in their

∗ Corresponding author. Tel.: +55 3491337459.E-mail address: estevaokienzan@yahoo.com.br (E. Alves-Silva).

ttp://dx.doi.org/10.1016/j.ecolind.2015.09.036470-160X/© 2015 Elsevier Ltd. All rights reserved.

bilateral symmetry (Santos et al., 2013), which tends to becomemore severe as the stressor becomes stronger (Cuevas-Reyes et al.,2011b).

In spite of the extensive literature concerning FA and envi-ronmental stressors (reviewed by Møller and Shykoff, 1999), fewstudies have addressed the relationship between FA and insectherbivory (see Cornelissen and Stiling, 2005; Santos et al., 2013;Telhado et al., 2010). These studies, however, show alternativelystrong, weak or no association at all between FA and insect her-bivory (Alves-Silva, 2012; Costa et al., 2013; Telhado et al., 2010).This is because the FA–herbivory literature (studies examiningthe relationship between FA and herbivory) is still relatively con-troversial and two lines of research can be recognized. The firstconsiders FA as a surrogate for herbivore attack, as stressed plantshave more free nitrogen and less defensive or secondary defensecompounds (Cornelissen and Stiling, 2005). Such an approach fol-

vory-induced stress: Leaf developmental instability is caused by015), http://dx.doi.org/10.1016/j.ecolind.2015.09.036

lows the concepts of the plant stress hypothesis (discussed in Møller,1995; see also Mattson and Haack, 1987; White, 1984). Conversely,other studies demonstrate that herbivores themselves might act asplant stressors, affecting the bilateral pattern of growth in leaves

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nd thus inducing FA (Santos et al., 2013; Zvereva et al., 1997b).his approach, still scarcely studied, will be hereafter named ashe herbivory-induced stress hypothesis for the sake of clarity. Theypothesis states that herbivore damage, either in young or mature

eaves, will affect the expected pattern of growth in bilaterally sym-etrical characters eventually giving rise to increased FA levels.

he purpose of this work was to investigate whether or not insecterbivores cause leaf FA.

Thrips (Insecta: Thysanoptera), a group of ubiquitous, sap-ucking small insects (Mound and Marullo, 1996) were chosen asur study herbivores. Aggregations of Pseudophilothrips obscuri-ornis Priesner, 1921 (Tubulifera: Phlaeothripidae) (hereafterseudophilothrips) are commonly found on Neotropical Malpighi-ceae (Alves-Silva and Del-Claro, 2014). Both adults and larvae feedn leaf buds (rudimentary and undeveloped foliage) and youngeaves, causing mature leaves to show distortions and numerousrown/dark necrosis marks. In general, herbivory by thrips influ-nces the nutrient balance as well as the photosynthetic rate of theirost plants, ultimately causing a loss in overall plant vigor and fit-ess (Cuda et al., 2008; De Borbón and Cardello, 2006; Mound andapater, 2003).

The system Pseudophilothrips – Malpighiaceae is ideal as a modelo examine whether herbivory by thrips causes leaf FA. To advancenowledge of FA–herbivory systems, study designs should be ableo examine unambiguously whether herbivores induce FA or ratherearch for asymmetric leaves to feed on (Díaz et al., 2004). In ourtudy system, thrips preferentially feed on leaf buds, thus FA onature leaves would be evidence that earlier herbivory by thrips

ncreased the developmental instability of leaves, which would bevidence for the herbivory-induced stress hypothesis.

Throughout the leaf flush season of the two study plants (seeelow), we examined the occurrence and feeding behavior of thripsn leaf buds. Further sampling and analyses of mature leaves aimedo evaluate the FA levels in uninjured and damaged leaves, i.e.he ones that were attacked by thrips during the leaf bud stage.

e hypothesized that the latter would display higher FA. Shouldhis hypothesis be corroborated, it might give support for theerbivory-induced stress hypothesis. We also examined the relation-hip between thrips abundance and leaf herbivory rates, and weredicted a positive relationship. To conclude, we compared thebundance of adults and larvae of Pseudophilothrips and we exam-ned their occurrence according to leaf developmental stage. Weypothesized that thrips, especially larvae (the stage where thrips

eed voraciously on plants), would be found feeding predominantlyn leaf buds and will contribute to FA of mature leaves.

. Material and methods

.1. Study system

Fieldwork was conducted in a Brazilian savanna area (230 ha;8◦59′ S–48◦18′ W) in Uberlândia city, Brazil, from January toovember 2011, and in March 2015 (see Alves-Silva and Del-laro, 2013; Vilela et al., 2014 for details on the study area). Thetudy plants, Banisteriopsis malifolia (Nees and Martius) B. Gatesnd Heteropterys escallonifolia A. Juss. are shrubs (<2 m high) withany branches. Leaves from both species have small trichomes dis-

ributed along the leaf blade on both sides, and a pair of extrafloralectaries occurs at the base near the petiole at each side. B. malifolia

eaf budding begins at the onset of the rainy season (October) andasts until early April. Leaves are smooth-margined, ovate, green-

Please cite this article in press as: Alves-Silva, E., Del-Claro, K., Herbiherbivore damage in early stages of leaf development. Ecol. Indicat. (2

sh and can reach up to 15 cm in length and 10 cm in width; thepex is acute and the base is rounded (Alves-Silva and Del-Claro,014). H. escallonifolia phenology is also markedly seasonal. Thislant produces leaves from late August to January, during the rainy

(rudimentary foliage – arrows). (b) An adult individual of P. obscuricornis. (c) Banis-teriopsis malifolia leaf, indicating how fluctuating asymmetry measurements wereperformed. Rs – right side; Ls – left side. Scale bars: a, c – 20 mm; b – 10 mm.

season. Its leaves are oblong, dark green and can reach up to 10 cmin length and 5 cm in width. The leaf apex is obtuse and the base isrounded (Urbanetz et al., 2013).

Thrips (Thysanoptera) are mostly known for their pest statusin many economically important crops (Kirk and Terry, 2003), butin fact, only about 2% of the ∼5000–6000 species are consideredpests, and belong to the suborder Terebrantia (Cavalleri et al.,2010; Monteiro et al., 2001; Morse and Hoddle, 2006; Mound andMarullo, 1996; Mound and Morris, 2007). Species within the sub-order Tubulifera (like Pseudophilothrips) are far less studied and arenot regarded as pests (Mound, 2002). Nonetheless, some species,like Liothrips are voracious herbivores, being under considerationfor biological control of invasive plant species (Cuda et al., 2008).

Pseudophilothrips is the sole herbivorous insect that feeds onmeristems, shoots, short-shoot leaves and leaf buds of B. malifo-lia and H. escallonifolia (Fig. 1a, b). Both adults (∼2 mm in length)and larvae (∼1 mm in length) are found in aggregations, but larvaeoccur predominantly on meristems, shoots and leaf buds, whereasadults are more mobile and are found especially on young leaves.Adults are black and winged, whereas larvae are wingless andreddish. Females lay eggs on lateral meristems, shoots and leafbuds.

2.2. Sampling

B. malifolia (n = 30) and H. escallonifolia (n = 48) were randomlychosen within the Brazilian savanna area. All individual plantsselected for this study belonged to the same population and werespread evenly in an area of ∼6 ha, and therefore, were prone tosimilar biotic and abiotic stresses (following Telhado et al., 2010).Shrubs were not shaded by the canopy of large trees and receiveddirect sunlight throughout the whole day. Plants were tagged priorto the leaf budding season, (B. malifolia in December and H. escal-lonifolia in August) and from each shrub, we selected the most apicalstem for FA and herbivory analyses (following Alves-Silva and Del-Claro, 2013), because variation in leaves from different parts ofplants might influence FA levels (Sibio and Rossi, 2012).

Pseudophilothrips individuals colonized B. malifolia and H. escal-lonifolia in January and September, respectively, at the first sign

vory-induced stress: Leaf developmental instability is caused by015), http://dx.doi.org/10.1016/j.ecolind.2015.09.036

of leaf budding. From the onset of leaf flush until leaf sampling,all other herbivores that might feed on leaves and affect the studywere removed from the study-tagged plants and were placed ondistant non-studied plants of the same species. Fortunately, all of

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he plants had few other folivores besides thrips, given that mosterbivores of Malpighiaceae feed on reproductive structures, andhe ones that feed on leaves are chewers (Alves-Silva et al., 2013;ächtold et al., 2013, 2014; Del-Claro et al., 1997; Reu and Del-Claro,005; Vilela et al., 2014). In addition, leaves damaged by thrips areot usually attacked by other herbivores (pers. obs.).

Plants were checked periodically (at an interval of 3–7 days) inhe field from tagging until leaf sampling in order to ensure thatll plants had the same phenological status (i.e. simultaneous leafudding and maturation of leaves) and hosted thrips, especially

arvae throughout the study period. Leaves from B. malifolia wereollected in mid-February 2011; by this time leaf buds attackedy thrips had become mature leaves and presented necrosis marksn the leaf blade. Fourteen leaves per individual plant, with equalumbers of injured and non-injured leaves, were sampled (n = 420

eaves) for further comparisons of FA. Mature leaves from H. escal-onifolia were collected in October 2011. A total of ten leaves, fivenjured and five uninjured per plant, were sampled (n = 480 leaves).

.3. Thrips abundance and herbivory

The abundance of thrips on plants (examined in 2011) was esti-ated once, just before leaf sampling, and only on stems where

eaves had been collected for herbivory and FA analyses. To examinehe feeding damage by Pseudophilothrips on the two Malpighiaceaepecies, collected leaves of each plant were photographed under

slide of transparent glass. The images were then transferred to computer and measurements of leaf necrosis area (mm2) wereerformed using the Image J software (adapted from Chen andilliams, 2006). Leaf area was also estimated and used to calcu-

ate the percentage of leaf necrosis area, which was achieved byividing the area of leaf damage by the leaf area (mm2).

In order to investigate whether thrips occurred predominantlyn leaf buds, young or mature leaves, we tagged 20 B. malifoliandividuals in early March 2015. At this point, the plant still had leafuds, young leaves and fully mature/developed leaves. The mostpical stem was selected from each individual plant and all thripsboth adult and larvae) were carefully observed and counted. Leafuds and leaves with thrips were collected, numbered for further

dentification and taken to the laboratory where their length waseasured (mm).

.4. Asymmetry measurements

Among the 420 leaves collected from B. malifolia, 18 of themresented large variations in the difference between left and rightlade widths and were discarded in subsequent analyses to avoidiasing the results (following Alves-Silva and Del-Claro, 2013; Costat al., 2013; Santos et al., 2013). In H. escallonifolia, 22 leaves werexcluded for the same reason. To assess leaf FA, the widths of alleaves were measured on both the right (Rs) and left sides (Ls),rom the leaf edge to the midrib, at the middle point of the leaf,hich corresponds to its widest part (following Berteaux et al.,

007; Costa et al., 2013; Ishino et al., 2011) (Fig. 1c). Leaves werehotographed under a transparent glass, and measurements were

ater made using the Image J software (adapted from Cornelissennd Stiling, 2005). All the digital images were calibrated to theearest 0.001 mm before measurements were taken, but final mea-urements were rounded to two decimal places for the sake oflarity.

.5. Measurement error

Please cite this article in press as: Alves-Silva, E., Del-Claro, K., Herbiherbivore damage in early stages of leaf development. Ecol. Indicat. (2

To test the accuracy of the measurements, a subsample of 49eaves of B. malifolia and 50 of H. escallonifolia was remeasurednd compared with the original Rs and Ls measurements. The

PRESSl Indicators xxx (2015) xxx–xxx 3

repeatability of measurements is mandatory in FA studies andindicates whether leaf sides and their asymmetries were mea-sured with sufficient precision to discard measurement errors(see Yezerinac et al., 1992). A two-factor analysis of variance(Anova) was used to determine whether the between-sides vari-ation was significantly larger than the measurement error (Santoset al., 2013). Plant individuals and leaf sides (Rs and Ls) wereused as variables. The variation in FA was greater than expectedby measurement error (B. malifolia F48,98 = 7.0538, P < 0.0001; H.escallonifolia F49,100 = 5.7737, P < 0.0001), indicating that leaf mea-surements were conducted with sufficient accuracy to discarderrors (Cuevas-Reyes et al., 2011a).

2.6. Assumptions of fluctuating asymmetry

According to Palmer and Strobeck (1986), it is necessary todiscriminate FA from other kinds of asymmetry. Fluctuating asym-metry refers to random and small deviations from symmetry inleaves, with a mean value of zero. Directional asymmetry (DA)depicts a scenario in which leaves have one side significantly largerthan the other, and the average differences of Rs minus Ls are alwaysgreater or less than zero. Antisymmetry (AS) is a lack of symmetry,with no specific direction where the values in a frequency charthave a bimodal distribution. To assure that our data fitted purelyFA and not other types of asymmetry, we conducted the follow-ing tests: (i) DA was examined by testing whether the average Rsminus Ls value differed from zero (one sample Student’s t-test); (ii)to check for antisymmetry, the normality of the Rs minus Ls dis-tribution was examined with the Lilliefors’ normality test. All ofthese tests were made at the leaf level (following Alves-Silva andDel-Claro, 2013; Santos et al., 2013).

Our data fit the assumptions of FA as there was no differ-ence in the mean of Rs minus Ls measurements, consequently DAwas discarded (B. malifolia: t403 = 1.3796, P > 0.05; H. escallonifolia:t457 = 0.0732, P > 0.05). Moreover, Rs minus Ls measurements alsodid not depart from normality, thus AS was rejected (Lilliefors’normality tests P > 0.05 in both plants). Therefore, in this studyFA was confirmed in both plant species. Absolute FA was calcu-lated as the mean difference between the right and left sides, i.e.,FA = [(�|(Rs − Ls)|/n], where n indicates the number of leaves mea-sured (Palmer and Strobeck, 1986; Santos et al., 2013). Since inour study FA was not related to leaf size (R2 < 1; P > 0.05 in bothMalpighiaceae species), the use of a size-corrected formula (as seenin Cornelissen and Stiling, 2005; Costa et al., 2013) was unneces-sary.

2.7. Statistical analyses

Quantitative data are presented as the mean ± standard error(SE). Non-parametric statistical tests were performed wheneverthe data did not satisfy the assumptions of the normal distribu-tion (normality test P < 0.05), and transformations were unableto achieve data normality. For the sake of clarity, figures showuntransformed data. The FA of healthy (no signs of herbivory) anddamaged leaves of B. malifolia and H. escallonifolia (log-transformeddata) was compared using two-sample Student’s t-tests. In theseanalyses, we used the mean FA values per plant (following Alves-Silva and Del-Claro, 2013; Cornelissen and Stiling, 2011; Santoset al., 2013). Differences in FA levels between H. escallonifoliaand B. malifolia; abundance of thrips (adult + larvae) on each plantspecies; and abundance of larvae and adult thrips (original datalog-transformed) were all compared using two-sample Student’s t

vory-induced stress: Leaf developmental instability is caused by015), http://dx.doi.org/10.1016/j.ecolind.2015.09.036

tests.The percentage of leaf damage was compared between plant

species using a Student’s t-test after arcsine transformation ofthe data. To examine the relationship between thrips abundance

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ged and uninjured leaves in two species of Malpighiaceae. Pseudophilothripserbivory increased leaf FA in both plant species (* and *** are equal to P < 0.05nd P < 0.0001 according to Student’s t-tests).

nd herbivory, we conducted linear regressions for both Malpighi-ceae species. In this test, we used the herbivory area (mm2).erbivory rate (%) and leaf necrosis area were strongly correlated

B. malifolia: F1,28 = 248.7629, R2 = 0.8923, P < 0.0001; H. escalloni-olia: F1,46 = 117.3321, R2 = 0.7184, P < 0.0001), thus, we decided tose to use only herbivory area (mm2) to avoid redundancy.

In order to examine the occurrence of thrips according to leafizes (analyzed in 2015), sampled leaves were ranked in four groupsccording to their size: shoots, leaves ≤10 mm in length; leavesetween 10.1 and 20 mm in length; and leaves >20 mm in length.hese categories describe all the leaves where thrips were founduring the sampling. The abundance of larvae and adult individ-als of Pseudophilothrips was compared using the Mann–Whitney

test, a non-parametric analysis for two-samples. The compar-son between the frequency of leaf categories sampled and therequency of thrips was made using a G test. The abundance of thripsn each leaf size category was compared using Kruskal–Wallis tests.tatistical procedures are in accordance with Zar (2010) and wereerformed using Systat 10 and GraphPad Prism 5.0 softwares.

. Results

In both plant species, FA levels were significantly higher inamaged leaves (B. malifolia t58 = 1.8646, P < 0.05; H. escallonifo-

ia t94 = 4.6350, P < 0.0001). The average difference in FA betweenon-injured and damaged leaves was 15% and 27% in B. malifoliand H. escallonifolia, respectively (Fig. 2). These results support theerbivory-induced stress hypothesis, because herbivory by thrips inarly stages of leaf development (leaf buds) gave rise to significantncreases in leaf FA of mature leaves. Between-species compari-on revealed that damaged leaves of B. malifolia were on average8% more asymmetrical than leaves of H. escallonifolia (t76 = 4.0087,

< 0.0001).Thrips were on average roughly 11% more abundant on stems of

. escallonifolia (12.06 ± 0.54 thrips per stem per plant) compared to. malifolia (10.83 ± 0.71), but this difference was not statisticallyignificant (t76 = 1.3876, P > 0.05). The abundance of thrips larvae,he stage where thrips feed voraciously on plants, was 1.9-fold and.05-fold higher than the abundance of adults in B. malifolia and H.scallonifolia, respectively (t58 = 3.4874, P > 0.001 and t94 = 10.2062,

> 0.0001) (Fig. 3).In B. malifolia, leaves damaged by thrips had necrosis

Please cite this article in press as: Alves-Silva, E., Del-Claro, K., Herbiherbivore damage in early stages of leaf development. Ecol. Indicat. (2

arks that covered on average 20.33 ± 5.46 mm2 of the blade,hich corresponds to 0.65 ± 0.14% of leaf area. In H. escal-

onifolia, necrosis occupied 17.58 ± 1.43 mm2 of the leaf blade1.12 ± 0.09% of leaf area). In both cases, the average herbivory

Fig. 3. Abundance (mean ± SE per stem/per plant) of larvae and adults of Pseu-dophilothrips obscuricornis in two Malpighiaceae species (** and *** equal to P < 0.001and P < 0.0001 according to Student’s t-tests).

by thrips affected roughly 1% of the leaf area (0.65 ± 0.14%and 1.12 ± 0.09% in B. malifolia and H. escallonifolia, respec-tively), being higher in H. escallonifolia (t74 = 4.8281, P < 0.0001).The relationship between Pseudophilothrips abundance and leafherbivory was positive and statistically significant in both B. mal-ifolia (F1,28 = 22.6301, R2 = 0.4470, P < 0.0001) and H. escallonifolia(F1,46 = 11.9082, R2 = 0.2056, P < 0.01) (Fig. 4a, b).

In 2015, a single stem of B. malifolia was examined in eachplant in order to examine whether thrips were encountered pre-dominantly on leaf buds and young leaves. A total of 106 leaves(5.3 ± 1.49 leaves per stem/per plant) and 15 leaf buds (0.75 ± 0.19leaf buds per stem/per plant) were sampled, and 76 thrips werefound. Both the frequency and the abundance of thrips were signif-icantly higher on leaf buds (Fig. 5, Table 1). Leaves >20 mm in lengthoccurred most frequently on stems, however thrips were foundmostly on leaf buds and on leaves ≤10 mm in length (G = 61.4525,df = 3, P < 0.0001) (Fig. 5).

Pseudophilothrips larvae were far more abundant than adults(n = 70, 0.58 ± 0.07 and n = 6, 0.05 ± 0.02, respectively, U = 4486.5;P < 0.0001). Adults were scarcely sampled, and found only onleaf buds and on leaves >20 mm in length (n = 2 and n = 4 indi-viduals, respectively). Larval abundance was higher on leaf buds(37%, n = 26; 1.73 ± 0.18) and leaves ≤10 mm in length (31%, n = 22;0.92 ± 0.13) (H = 4694.92; df = 3, P < 0.0001) (Table 1). The meanabundance of larvae on leaf buds was on average 15-fold higherthan on leaves >20 mm in length (Table 1).

4. Discussion

Herbivory by Pseudophilothrips caused stress to B. malifolia andH. escallonifolia, which was reflected in increased FA in leaves,corroborating the herbivory-induced stress hypothesis. The featuresof our study system permit us to make three observations sup-porting the herbivory-induced stress hypothesis. First, thrips wereobserved to feed especially on leaf buds. Subsequently, matureleaves presented the classical symptoms of thrips feeding, such asnecrosis marks on the leaf blade (see Chen and Williams, 2006).In addition, mature leaves also presented significant incrementsin FA. Second, according to the literature (Mound, 2005; Moundand Marullo, 1996; Pearsall, 2000; Tamo et al., 1993) thrips larvae(including tubuliferan members – Cuda et al., 1999, 2008) inflictthe greatest damage to the plant, because they feed and aggregatefor long periods on meristems and shoots until pupation, which

vory-induced stress: Leaf developmental instability is caused by015), http://dx.doi.org/10.1016/j.ecolind.2015.09.036

occurs on the soil. In our study, larvae were far more abundant thanadults, and were found predominantly on leaf buds. Therefore, boththe natural history of larval stages and the timing of leaf damagewere responsible for increments in the FA of mature leaves. Third,

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Fig. 4. Relationship between herbivory (mm2) and Pseudophilothrips abundance in (a) Banisteriopsis malifolia and (b) Heteropterys escallonifolia. In both plants herbivory wassignificantly and positively related to thrips herbivory.

Table 1Abundance of Pseudophilothrips obscuricornis according to different leaf categories. Please check the text for appropriate statistical analyses.

Leaf categories Thrips (larvae + adults) Larvae only

Mean ± SE Median (range) Mean ± SE Median (range)

Shoots 2.00 ± 0.24 2 (1–4) 1.73 ± 0.18 2 (1–3)–2)

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Leaves <10 mm 0.92 ± 0.13 1 (0Leaves 10–20 mm 0.48 ± 0.11 0 (0Leaves >20 mm 0.20 ± 0.07 0 (0

enerally high levels of stress are expected in plants attacked byerbivores (Puerta-Pinero et al., 2008; Zvereva et al., 1997b). Stressccurs because feeding damage alters plant metabolism and influ-nces plant development, architecture and performance (Loudat al., 1990; Karban and Strauss, 1993). Since in some cases her-ivory can have a detrimental effect on plant fitness (Cuda et al.,999, 2008; Marquis, 1984; Mauricio et al., 1993), it is not sur-rising at all to find that FA can also arise from herbivore feedingamage. Unfortunately, only a few studies have examined causalelationships between leaf herbivory and its effects on FA (Olofssonnd Strengbom, 2000; Zvereva et al., 1997b).

Despite the large insect herbivore fauna associated withalpighiaceae in the Brazilian savanna (Vilela et al., 2014), shoots,

eaf buds and young leaves of this botanical family are exclusivelyttacked by thrips. Necrosis affecting the whole leaf blade is uncom-on, but thrips-attacked leaves usually become twisted, crispy

nd sclerotized (Alves-Silva and Del-Claro, 2014). Taking all thisnto consideration, and the fact that in our study, damaged leaves

Please cite this article in press as: Alves-Silva, E., Del-Claro, K., Herbiherbivore damage in early stages of leaf development. Ecol. Indicat. (2

resented significant increases in FA, we can assume that Pseu-ophilothrips are important stressing agents to both B. malifolia and. escallonifolia. Interestingly, herbivory by thrips affected barelyore than 1% of the leaf blade. Even so, thrips-attacked leaves were

ig. 5. Occurrence of thrips according to the frequency of leaves and shoots exam-ned in Banisteriopsis malifolia.

0.92 ± 0.13 1 (0–2)0.48 ± 0.11 0 (0–2)0.12 ± 0.06 0 (0–2)

15% and 27% more asymmetrical than uninjured leaves of B. mali-folia and H. escallonifolia, respectively. To date, studies have shownthat FA arises from the feeding behavior of insects that feed onlarge portions of the leaf blade, such as chewers and defoliators(Zvereva et al., 1997b). Therefore, our results are surprising becauseroughly 1% of leaf damage in Malpighiaceae was sufficient to causesignificant increments in FA. By means of comparison, even heavybrowsing by mammalian herbivores is not enough to inflict FA inCanadian trees (Berteaux et al., 2007).

Both B. malifolia and H. escallonifolia showed some levels of FAregardless of thrips herbivory, as shown in uninjured leaves. Thisresult is entirely expected, because plants in nature are exposedto several and (sometimes) unpredictable environmental distur-bances (e.g., wind, sunlight exposure, soil nutrients, humidity,pollution), which can affect their development and homeostasis,eventually causing FA in leaves (Alves-Silva and Del-Claro, 2013;Cornelissen and Stiling, 2010; Cuevas-Reyes et al., 2011a; Kozlovet al., 1996; Puerta-Pinero et al., 2008). One of our main findingswas that thrips herbivory boosted the leaf FA levels, indicatingthat besides environmental stressing agents, a biotic factor wasalso responsible for increased stress levels in two different plantspecies (Díaz et al., 2004). In this context, one could assume thatthrips were attracted to previously stressed plants, and that leaveswere already asymmetrical before herbivore damage (Cornelissenand Stiling, 2005). Nonetheless, as our data have shown, thripsoccurred predominantly on leaf buds and undeveloped leaves, sothe herbivory damage occurred regardless of structural symmetryand before leaves presented clear signs of bilateral symmetry. Ina similar approach, Santos et al. (2013) found that parasitism bygalls also occurred in the early stages of leaf development, whenno pattern of leaf symmetry could be perceived and parasitism ofmature leaves was positively related to increases in FA.

5. Conclusion

vory-induced stress: Leaf developmental instability is caused by015), http://dx.doi.org/10.1016/j.ecolind.2015.09.036

In this study, we found support for the herbivory-induced stresshypothesis, as leaf buds damaged by thrips showed high levels ofFA when mature. This study is one of the few that demonstratesa significant relationship between herbivory and FA, especially

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n the Neotropics. To the best of our knowledge, only one studyas examined the relationship between a sucker herbivore and FAo date (Telhado et al., 2010). We believe that novel approachessing sucker herbivores (e.g., aphids and membracids) and theirelationship with leaf FA might reveal meaningful results, becauseembers of this guild spend a considerable time feeding on leaves

nd shoots, causing chemical and nutritional changes in the hostlant (Burd and Burton, 1992; Heng-Moss et al., 2003; Telang et al.,999). In this context, an appreciation of how and to what extenterbivores affect the developmental instability of their host plantsight be an important tool for the advance of FA–herbivory stud-

es, especially in the Neotropics, where FA studies are still scarce inomparison to those in temperate regions (Telhado et al., 2010).

cknowledgements

We are grateful to the staff of the Clube de Cac a e Pesca Itororóe Uberlândia, where the fieldwork was carried out; the stafff Herbarium Uberlandensis for their kindness in showing plantpecies; three anonymous reviewers for their polite commentsnd suggestions, which increased the quality of the manuscript;apes (Coordenac ão de Aperfeic oamento de Pessoal de Nível Supe-ior) and CNPq (Conselho Nacional de Desenvolvimento Científico

Tecnológico) for funding. The authors have no conflict of interesthatsoever.

ppendix A. Supplementary data

Supplementary material related to this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.ecolind.2015.09.36.

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