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Received: 12 February 2021    Accepted: 23 March 2021

DOI: 10.1111/1365-2745.13659

RESEARCH ARTICLE

From the leaf to the community: Distinct dimensions of

phytochemical diversity shape insect–­plant interactions within
and among individual plants

Leandro G. Cosmo1  | Lydia F. Yamaguchi2  | Gabriel M. F. Felix1  |

Massuo J. Kato2  | Rodrigo Cogni3  | Martín Pareja4

1
Programa de Pós-­G raduação em Ecologia,
Institute of Biology, University of Campinas
–­UNICAMP, Campinas, Brazil
2
Department of Fundamental Chemistry,
Institute of Chemistry, University of São
Paulo, São Paulo-­SP, Brazil
3
Department of Ecology, University of São
Paulo, São Paulo, Brazil
4
Department of Animal Biology, Institute
of Biology, University of Campinas –­
UNICAMP, Campinas, Brazil
Correspondence
Martín Pareja
Email: mpareja@unicamp.br
Present address
Leandro G. Cosmo, Ecology Graduate
School, Biosciences Institute, University of
São Paulo, São Paulo, 05508-­900, Brazil
Funding information
FAPESP, Grant/Award Number:
2014/50316-­7, 2013/25991-­0, 2019/22146-­
and 3; CAPES, Grant/Award Number: 001;
FAEPEX-­UNICAMP, Grant/Award Number:
60412 and PAPDIC 2014/4715; CNPq,
Grant/Award Number: 304607/2019-­3,
307015/2015-­7 and 307447/2018-­9; Royal
Society
Handling Editor: Tobias Züst
Abstract
1. Plant secondary chemistry is known to be an important driver of plant–­insect
community structure across ecological scales. Recently, the concept of phyto-
chemical diversity (PD) has been introduced to help describe variation in plant
secondary chemistry and explain how this variation affects community structure.
Previous studies show that PD among individuals and species results in phyto-
chemical mosaics, known as the phytochemical landscape. However, plant traits
can vary at finer scales, such as within individuals, and even a single host plant may
be perceived as an entire phytochemical landscape by an interacting insect.
2. Using the neotropical shrub Piper amalago, we tested and compared how her-
bivory, caterpillar biodiversity and plant–­herbivore network structure are affected
by the compositional (number and concentration of compounds) and structural
(diversity of distinct chemical structures) dimensions of PD. We analysed variation
among individual plants and among-­plant height strata within individual plants.
This allowed us to decompose PD within and among plant individuals and analyse
how variation at both scales affects the plant–­herbivore network.
3. We found that both within and among plants greater structural diversity de-
creased herbivore feeding damage. Furthermore, each dimension of PD has dif-
ferent effects on herbivore biodiversity and network structure depending on the
scale of biological organization.
4. Within plants, the compositional dimension, specifically low concentrations of
compounds tentatively identified as Piper amides, increased herbivore biodiver-
sity. This dimension also increased the capacity of strata within plants to mediate
ecological cascades through direct and indirect effects on herbivore abundance
in the plant–­herbivore network. In contrast, a greater structural diversity among
plants decreased herbivore biodiversity and the capacity of plants to affect all
other herbivores and plants directly and indirectly in the network.
5. Synthesis. Based on our results we expand the concept of the phytochemical
landscape to multiple scales of biological organization and provide evidence that
PD may be maintained by how its multiple dimensions have distinct roles across
scales of biological organization.

Journal of Ecology. 2021;109:2475–2487. wileyonlinelibrary.com/journal/jec© 2021 British Ecological Society     2475 |

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KEYWORDS
chemodiversity, compositional diversity, herbivory, phytochemical landscape, phytochemical
variation, plant defence, plant–­herbivore interactions, structural diversity
1 |  I NTRO D U C TI O N
Understanding how plant secondary chemistry and plant diversity
affect community structure has been a major driver of ecological
and evolutionary research over the last 50  years (Becerra,  2015;
Ehrlich & Raven, 1964; Tilman et al., 1996). Although interest in vari-
ation in plant chemistry has a long history, recently chemical varia-
tion and biodiversity have been integrated through the concept of
phytochemical diversity (PD) and its cascading ecological effects
(Denno & McClure, 1983; Iason et al., 2005; Kessler & Kalske, 2018;
Moore et  al.,  2014; Randlkofer et  al.,  2010). Answering the ques-
tion of how PD affects insect communities remains challenging be-
cause it involves disentangling two major components of PD. First,
PD is multidimensional. Phytochemical blends consist of many com-
pounds that differ in their concentration. The richness and abun-
dance of compounds establish a compositional dimension of PD.
Each compound, in turn, has a unique structure, composed of dis-
tinct chemical structural features (e.g. a methyl, phenol, amine and
other groups) that are associated with the biological activity of com-
pounds (Gao et al., 2018; Richards et al., 2018). Similar to the com-
positional dimension, the richness and abundance of these structural
features both within and among compounds constitute a structural
dimension of PD. Consequently, phytochemical blends can have a
high compositional diversity, but a low structural diversity and vice
versa (Figure 1). Thus, disentangling how each dimension of PD af-
fects plant–­insect interactions is a key step towards understanding
the processes that maintain and drive the evolution of plant chem-
ical traits and patterns of herbivore diversity and herbivory (Dyer
et al., 2003; Glassmire et al., 2019; Richards et al., 2010, 2012, 2016;
Wetzel & Whitehead, 2020). Second, analogous to species diversity,
PD can vary across different scales of biological organization, from
the individual plant level to the community (Kessler & Kalske, 2018;
Moore et  al.,  2014). At each scale, PD may distinctly shape herbi-
vore biodiversity and patterns of herbivory (Glassmire et al., 2019;
Hunter,  2016; Massad et  al.,  2017). Therefore a major challenge is
to disentangle how the multiple dimensions of PD vary and affect
herbivore biodiversity at different scales of biological organization
(Kessler & Kalske, 2018; Wetzel & Whitehead, 2020).
Over the last few years, several studies have begun to address
this challenge. At the species level, Richards et al. (2015) measured
1
PD based on the number of peaks detected in H nuclear magnetic
resonance (NMR) spectra, which gives a measure of the number
of different structural features present in a blend (Figure  1a). This
approach captured, to a reasonable extent, the structural dimen-
sion of PD, which was found to increase herbivore diversity and
decrease herbivory (Richards et  al.,  2015). At the individual level,
increased variation in the foliar glucosinolate profiles of Brassica ol-
eracea chemotypes, hence a compositional dimension, increased the
COSMO et al.
diversity of insect herbivores and reduced the resulting damage suf-
fered by the plants (Bustos-­Segura et al., 2017; Poelman et al., 2009).
Likewise, at this same level of organization, Glassmire et al. (2016,
2019) found that both the compositional and structural dimensions
of PD increase along elevational gradients and with exposure to UV
light, which, in turn, reduces caterpillar diversity and can affect the
genetic structure of specialist insects. Therefore, among plant spe-
cies and individuals, the structural and compositional dimensions
can produce phytochemical mosaics, recently envisioned as a phy-
tochemical landscape, which can interact with herbivorous insect
performance and have community-­wide consequences (Glassmire
et al., 2019; Hunter, 2016).
Insects, however, can respond to small variation in plant chem-
ical traits, and herbivore performance may be affected even when
leaf quality differs within a single host plant (McCormick et al., 2012;
Pareja et  al.,  2009; Wetzel et  al.,  2016). Such ‘fine-­grained’ varia-
tion is widespread in nature, and leaf morphology, phenolic content,
flower nectar content, fruit and seed mass have all been shown to
vary within plants (Herrera,  2017; Herrera et  al.,  2015; Wetzel &
Meek, 2019). Hence, from an insect's perspective, a single individ-
ual plant may constitute an entire landscape of traits and when PD
varies within plants this can be perceived as an entire, fine-­grained,
phytochemical landscape (Herrera,  2009). This would suggest that
the phytochemical landscape consists of several layers across scales
which can be perceived by herbivorous insects and give rise to com-
plex feedbacks through different biological scales. Thus, to under-
stand such feedbacks, it is essential to assess and compare how the
many dimensions of PD shape herbivore biodiversity and herbivore
damage at different scales of biological organization, from the leaf to
the ecosystem (Levin, 1992; Wetzel & Whitehead, 2020).
In order to better understand how different dimensions of PD
shape insect–­plant interactions across the levels of biological organi-
zation, we performed a field study in a Brazilian tropical forest using
the shrub Piper amalago and its caterpillar herbivores. We quanti-
fied the structural and compositional dimensions of PD within and
among plants and its effects on herbivory, caterpillar biodiversity
and network structure. Specifically, our aims were (a) to address how
much variation in PD, caterpillar biodiversity and herbivory occurs
within plants and among plants; and (b) to test whether the struc-
tural and compositional dimensions of PD within and among indi-
vidual plants affect herbivore biodiversity, network structure and
herbivory. Addressing these aims will strengthen our understanding
of PD by separating the effects of composition (compound number
and identity) and structural diversity. Furthermore, focussing on the
effects of PD on ecological networks through two different scales
of biological organization (within and among plants) will allow us to
begin to understand how effects of PD can cascade through the
community and through ecological scales.
COSMO et al. Journal of Ecology|
      2477

F I G U R E 1   Hypothetical illustration of how the different dimensions of phytochemical diversity can be expressed in plants. (a) Five
different naturally occurring compounds with different structures, which can be reasonably captured by peaks in a 1H NMR spectra. In
an 1H NMR, each peak corresponds to the hydrogen signals present in compounds. These hydrogens resonate with different frequencies
depending on their chemical environment (e.g. a methyl group), which results in different peaks along the spectra. (b) Hypothetical scenarios
of how phytochemical blends can differ in their compositional and structural diversity, and illustrations of how they can be quantified with
the resulting high-­performance liquid chromatograms (HPLC) and 1H NMR spectra of the mixture. In an HPLC, each peak corresponds to at
least one compound that was separated from the mixture. The combinations shown are not exhaustive and there are many ways through
which phytochemical blends can differ in their structural and compositional diversity (e.g. many simple compounds with non-­redundant
structural features can also result in a high compositional and structural phytochemical diversity). The height, shape and position of peaks in
the LC are arbitrary and merely illustrative. 1H NMR spectra presented are not actual spectra but simulations predicted from the structure of
each compound and the hypothetical phytochemical blends using the MestReNova software (version 14.2.0)

2 | M ATE R I A L S A N D M E TH O DS
2.1 | Study site and system
This study was carried out in a semi-­deciduous tropical forest at the
Reserva Biológica Municipal da Serra do Japi (Jundiaí, São Paulo,
Brazil, 23°14'S 46°58'W; see Supporting Information for details). We
used the shrub P. amalago L. (Piperales: Piperaceae) and its herbivo-
rous caterpillars as a model system. Piper amalago is an Atlantic Forest
perennial shrub that grows between 1 and 7  m in height (Cosmo
et al., 2019; Guimarães & Valente, 2001). The genus Piper contains
about 1,000 species and is abundant in the Neotropics (Dyer &
Palmer, 2004). Piper species leaves are rich in chemical compounds,
which have been found to decrease herbivory and increase herbi-
vore species and genetic diversity (Glassmire et al., 2016; Richards
et al., 2015). Caterpillars (especially of the family Geometridae) are
the main Piper herbivores (Cosmo et al., 2019; Dyer & Palmer, 2004)
and are often dominant in herbivore communities. They have low
dispersal capabilities among plants but can change their feeding be-
haviour within individual plants (Novotny et al., 2006; Pogue, 2009;
Singer, 2016). Therefore, Piper species and their herbivorous cater-
pillars are excellent models for phytochemical and ecological studies
in the tropics (Dyer & Palmer, 2004).
2.2 | Sampling, data collection and processing
2.2.1 | Plant sampling
We sampled individual, naturally growing P. amalago plants at the
study site. At the beginning of the study, we marked all plants
found along three trails in the study site (the trails had a combined
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Journal of Ecology
extension of approximately 6.7  km), that were at least 1  m apart
from each other with a total height of at least 30 cm. In total, we
marked 128 plants. To control for seasonal effects, we randomly
sampled plants at four different times for 1  year, and each plant
was sampled only once (once sampled, the plant was removed from
the future sampling pool). We sampled 32 individuals in July 2017,
and another 32 individuals in October 2017. Because four of the
remaining 64 marked individuals died, crushed by trees falling dur-
ing the rainy season after October 2017, we sampled 30 individuals
in January 2018 and another 30 individuals in April 2018. Thus, we
collected data for 62 different individuals in the dry season (July
2017 and April 2018) and another 62 individuals in the rainy season
(October 2017 and January 2018), totalling 124 individuals for the
entire study period. We measured plant total height and total num-
ber of leaves to control for plant size and proximity to the canopy,
which can affect herbivore biodiversity and herbivory (Glassmire
et al., 2019). Each plant was divided into three strata of equal height,
starting at the first branch with leaves. Stratum height was defined
as: SH = TH − IH ∕3, in which SH is the height of each stratum, TH is
( )
the total height of the plant and IH is the height from the ground to
the first branch with leaves. Categorically, each stratum along the
plant vertical axis was defined as low, medium and high stratum. We
randomly collected 10 leaves within each stratum of each sampled
plant (30 leaves per individual plant). These strata within individual
plants were used as sampling units, totalling 372 units (three strata
from each of the 124 individual plants).
2.2.2 | Caterpillar biodiversity and network structure
To assess caterpillar biodiversity among and within plants and the
plant–­caterpillar network structure, we manually collected all cater-
pillars found within each stratum of the sampled plants. In the field,
caterpillars were initially identified as morphospecies and their total
count in each stratum was recorded. The caterpillars collected were
taken to the laboratory and reared in plastic pots following standard
techniques (Cosmo et al., 2014).
We characterized caterpillar biodiversity with three common
quantitative metrics: abundance (total counts), species richness and
species diversity, calculated as the Hill number of the Shannon–­
Wiener diversity index (from here on referred to as the caterpillar
SW index; Jost, 2006; Oksanen et al., 2019). A high Shannon–­Wiener
diversity indicates samples consisting of a greater number of more
evenly abundant caterpillar species, while a low value of the index
indicates either a smaller richness of caterpillar species and/or a
highly uneven distribution of abundances. For simplicity, the combi-
nation of these three metrics will be referred to as caterpillar biodi-
versity throughout the text.
Next, we used tools from network analysis to understand how PD
affects the capacity of each plant to propagate direct and indirect ef-
fects in the plant–­caterpillar network. We constructed a binary qualita-
tive and a weighted quantitative bipartite interaction network in which
each plant stratum (our sampling unit) is linked to a caterpillar species.
COSMO et al.
In the binary network, links represent the presence of a specific inter-
action between plant strata and caterpillar species, without consider-
ing the intensity of the interaction (the links do not have weights). In
the weighted network, we used the abundance of caterpillars in each
stratum as link weights. Thus, the binary network allows us to under-
stand the capacity of each plant or stratum to propagate direct and
indirect effects through the identity of herbivores, and in the weighted
network the role of each stratum is mediated by both herbivore iden-
tity and abundance. In both the binary and weighted networks, we
measured the capacity of each plant stratum to propagate direct and
indirect effects through three node-­level metrics: degree, closeness
centrality and Katz centrality. The degree quantifies the total number
(in the binary web) or total weights (in the quantitative web) of direct
interactions of a given node. Closeness centrality (Freeman,  1978)
measures the average shortest distance of a node to all others in
the network and thus, how quickly and efficiently a node can reach
all other nodes through either direct and/or indirect paths. Shortest
paths were identified via the igraph package (Csardi & Nepusz, 2006)
and in the weighted network we used the inverse of link weights as
the cost of a given path (i.e. higher abundance indicates that a given
path has a low cost). Katz centrality (Katz 1953) quantifies the amount
of direct and indirect pathways of all lengths that connects to a node
in a network. Hence, in contrast to closeness centrality that uses only
the shortest paths, Katz centrality measures the capacity of each node
to reach all others through pathways of all lengths. These three met-
rics, therefore, allow us to quantify the capacity of each plant stratum
to directly affect herbivores (degree), directly and indirectly affect
other herbivores and plant strata through shorter and efficient paths
(closeness centrality) and affect the entire network through all possible
direct and indirect pathways (Katz centrality). All analyses were per-
formed in the R environment v 3.6.0 (R Core Team, 2019).
2.2.3 | Leaf area and leaf damage measurements
To estimate leaf area and leaf damage, we photographed all collected
leaves against a white background along with a fixed-­length meas-
urement reference scale and used the software ImageJ to measure
leaf area (in cm2) from the images. This same software was used to
reconstruct leaves that had chewing herbivore damage and estimate
the total leaf area before herbivore damage. We then used the re-
constructed leaf area to calculate the proportion of total leaf area
lost to chewing herbivores. Finally, to estimate herbivory within and
among individual plants we used the average loss of leaf area of the
10 collected leaves for each stratum and the 30 collected leaves for
each individual plant respectively.
2.2.4 | Structural and compositional dimensions
of PD
To assess the structural and compositional dimensions of PD among
and within individual plants, we analysed the phytochemical profiles
COSMO et al.
of crude methanol extracts of the same leaves sampled to assess
1
herbivory. We used two complementary analyses: H NMR spectros-
copy and high-­performance liquid chromatography–­high-­resolution
electrospray ionization mass spectrometry (HPLC-­HRESIMS).
We summarized the structural dimension of PD through the
1
processed H NMR spectra (full description provided in Supporting
Information). The structural dimension was employed here to de-
fine the variety in the number of the structures and/or structural
complexity that was represented by the number and intensity of
hydrogen signals (peaks) in the NMR spectra. Following previous
studies, as a measure of the structural dimension of PD, we used
Hill's number of the Shannon–­Wiener diversity index for each plant
sample, calculated from the number of peaks and the values of
their integrated areas (Glassmire et  al.,  2019; Jost,  2006; Richards
et  al.,  2015; Wetzel & Whitehead,  2020) in the vegan package
(Oksanen et al., 2019). In 1H NMR spectra, each peak represents the
signal of hydrogen-­1 nuclei that resonate at different frequencies de-
pending on their chemical environment. These peaks are associated
with unique structural features of compounds (e.g. a methyl group),
and redundant structural features can give rise to peaks in the same
range of frequencies. Thus, the number of peaks in an NMR spec-
trum can be associated with the richness of structural features of the
compounds present in phytochemical blends. The relative intensity
of each peak in the spectra is proportional to the frequency of occur-
rence of the structural feature in a given molecule. Calculating the
1
Shannon–­Wiener diversity from H NMR peaks allows us to quantify
the structural diversity of phytochemical blends in terms of the rich-
ness and intensity (evenness) of structural features. Analogous to
species diversity, high values of structural diversity represent phy-
tochemical blends whose 1H NMR spectra show a high richness of
structural features (many peaks) whose abundance (signal intensity)
is very even. Such high values can be a result, for instance, of a single
(or few) compound with many non-­redundant structural features, or
many simpler compounds that do not share the same structural fea-
tures (Figure 1).
The compositional dimension of PD was measured through
HPLC-­HRESIMS processed data (full description provided in SI).
The processed peak areas from the total ion chromatograms were
integrated, adjusted by sample dry weight, normalized into propor-
tions, clr-­transformed (Brückner & Heethoff, 2017) and submitted to
PCA. The first and second PCs (hereinafter referred to as comp-­PC1
and comp-­PC2) explained approximately 78% of the total variation
(Table S2) and were used to represent the compositional dimension.
In contrast to the 1H NMR spectra, this approach allowed us to as-
sess the compositional dimension of PD and its effects on caterpil-
lar biodiversity in terms of the identity of combinations of different
compounds in a chemical extract, rather than in terms of structural
features. Although metabolite identification in complex matrices is
a current major challenge and we could not reliably identify these
compounds, for some we provide their molecular formulae, which
brings some insight into comp-­PCs 1 and 2 (Table  S1). The two
principal components were highly negatively correlated with com-
pounds tentatively identified as Piper amides (Table S1, Domínguez
Journal of Ecology|
      2479
et al., 1985). For instance, comp-­PC1 had high negative loadings with
a compound tentatively identified as the major amide of P. amalago,
nigrinodine (Jacobs et al., 1999; Yamaguchi et al., 2011), and another
common Piper amide (2-­methoxy-­4,5-­methylenedioxy-­trans-­cinna
moyl pyrrolidine; Domínguez et al., 1985; Nascimento et al., 2012;
Shah et  al.,  1986) had a high negative loading for both comp-­PC1
and comp-­PC2. Both principal components also had high positive
loadings with other unidentified compounds.
2.2.5 | Decomposing phytochemical diversity into
within-­and among-­plant components
We decomposed our measurements of the structural and compo-
sitional dimensions of PD into their within-­ and among-­plant com-
ponents. This was a necessary step to avoid incorrect inferences of
effects that occur at the among-­group level to the within-­subject
level (the ‘ecological fallacy’, see Supporting Information for details).
For this we subtracted the means of these variables for individual
plants from each observation, a procedure known as within-­subject
centring, and a common method used to distinguish within-­ and
among-­subject effects of predictors (van de Pol & Wright,  2009).
From this we obtained new variables that vary only within plants
that were used together with individual plant means as predictors in
our statistical analysis.
2.3 | Hypotheses testing
To address our objectives, we divided our hypothesis testing into
three steps. First, we estimated the proportion of variation in our
data that occurs only among individual plants. These estimates were
calculated through the intra-­class correlation (ICC). The ICC uses the
mixed model framework to partition variance into its within and be-
tween grouping levels components. Such partitioning can be calcu-
lated as the ratio:
VG
ICC = ,
VG + VR
where VG is the among-­group variance and VR is the sample-­level re-
sidual variance in a mixed model fitted with only a random (grouping)
effect as predictor. Hence 1 –­ICC accounts for the variation contained
only within groups. We estimated the ICC through the r package rptR
(Stoffel et al., 2017), which uses a bootstrap resampling approach to
calculate uncertainty around ICC estimates (1,000 bootstraps were
used). Caterpillar abundance and richness were both fitted with a
Poisson GLMM, while Linear Mixed Models (LMM) were used for the
SW index and herbivory, as well as for our measures of the structural
and compositional dimensions of PD. For all these variables, individual
plant ID was used as a grouping effect.
Second, we tested the hypothesis that the structural and compo-
sitional dimensions of PD shape the capacity of each plant stratum
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Journal of Ecology
to directly and indirectly affect the entire plant–­caterpillar network.
We fitted LMMs (nlme package, Pinheiro et al., 2020) with the binary
and weighted versions of degree, closeness and Katz centrality as
response variables for the subset of plant strata which had at least
one interaction. In both LMMs, we used plant ID as a random effect.
In mixed models with a grouping random effect (e.g. individual plant
ID), including a group-­level predictor automatically tests for the
effects of this predictor on the group-­level means of the response
variable (Pinheiro et  al.,  2020; van de Pol & Wright,  2009). Thus,
combining mixed models with our within-­subject centring approach
allowed us to simultaneously test for the within-­ and among-­plant
effects of structural and compositional dimensions of PD in our re-
sponse variables. As explanatory variables, we used the within-­plant
and individual plant structural and compositional (comp-­PCs 1 and
2) dimensions, within-­plant strata, the individual plant height, season
and total number of leaves. All predictors were scaled prior to model
fitting to standardize coefficients in terms of standard deviations of
the mean xi−x . We tested the significance of predictors with Wald's
( )
𝜎x
type II test, and model assumptions were visually assessed through
the residuals.
Finally, we tested whether within and among plants the struc-
tural and compositional dimensions of PD shape caterpillar bio-
diversity and herbivory. For this, we used piecewise Structural
Equation Modelling (piecewiseSEM, Lefcheck,  2016), which is
a powerful approach to evaluate causal and cascading effects
among variables. We built a SEM in which we modelled each re-
sponse variable with LMMs or GLMMs (nlme package, Pinheiro
et  al.,  2020). Based on previous work with Piper insect–­p lant in-
teractions, we hypothesized the following causal relationships:
(a) individual plant height affects individual plant PD, which, in
turn, drives caterpillar biodiversity and herbivory; (b) within-­p lant
strata (from low to high) affect within-­p lant PD which also drives
caterpillar biodiversity and herbivory; (c) herbivory is affected by
caterpillar species diversity; (d) caterpillar abundance affects spe-
cies richness and both drive species diversity; (e) structural and
compositional dimensions of PD are correlated, both within and
among plants (in SEMs this can be specified as correlated errors);
and (f) individual plant height and within-­p lant strata also directly
affect caterpillar biodiversity and herbivory. Because caterpillar
biodiversity and herbivory in P. amalago is known to vary sea-
sonally and with host-­p lant leaf numbers (Cosmo et al., 2019), we
additionally included season (dry and rainy) and the total num-
ber of leaves on the plant as fixed effects for all response vari-
ables. Furthermore, plant ID was included as a random effect in
all models. Percentage herbivory was logit transformed (Warton
& Hui,  2011). Poisson error distributions were used for caterpil-
lar abundance and species richness, while Gaussian errors were
used for the remaining variables. Except for abundance and rich-
ness (because Poisson GLMM accepts only positive integers as
response variables), all predictors were scaled prior to model fit-
ting to standardize path coefficients in terms of standard devia-
tions xi−x . We assessed model goodness-­of-­f it through tests of
( )
𝜎x
directed separation (Lefcheck, 2016). This test evaluates whether
COSMO et al.
there are missing paths in the specified model and its significance
is assessed with Fisher's C statistic. Our initial model was a good
fit (Fisher's C = 28.9, p = 0.99, df = 50) and was the one chosen
as best model. All model assumptions were verified through the
package DHARMa (Hartig, 2017).
3 | R E S U LT S
3.1 | Caterpillar biodiversity, herbivory and the
dimensions of phytochemical diversity vary within
plants
In total, we collected 273 caterpillars, and, on average, each plant
harboured 2.2  ±  3.85 (mean  ±  SD) caterpillars, while we found
0.76 ± 1.47, 0.50 ± 1.01 and 0.97 ± 1.99 (mean ± SD) in the low, me-
dium and high strata respectively (Figure 2a). Caterpillar abundance
varied seasonally and was substantially higher in the dry season than
in the rainy season (Figure  2a). These caterpillars were distributed
among 18 morphospecies and two morphospecies of Eois (Piper spe-
cialists) were the most abundant and accounted for 86.8% of the
total (Table  S2). The proportion of leaf area lost to herbivory was
0.12 ± 0.08, varying from 0.00 to 0.48 and the low, medium and high
strata suffered 0.14 ± 0.08, 0.12 ± 0.08 and 0.11 ± 0.07 (mean ± SD)
respectively (Figure 2b).
Our variance partitioning through the intra-­class correlation
(ICC) revealed that both caterpillar biodiversity and the two dimen-
sions of PD differed in how much they varied within and among
plants (Figure  3; Table  S3). Caterpillar SW index, herbivory and
comp-­PC1, all varied substantially more within plants than among
plants. Richness varied more within plants, but confidence intervals
were wide. On the other hand, caterpillar abundance and the struc-
tural dimension of PD varied more among plants, while comp-­PC2
varied within plants as much as among plants.
3.2 | PD shapes the direct and indirect influence of
plant strata on the plant–­caterpillar network
When herbivores interact with different plants and plant strata,
these interactions can be depicted as networks. In the network, al-
though plants or plant strata do not directly interact, they can in-
directly affect each other through shared herbivores. For instance,
one plant may affect one or more herbivores, which in turn affects
other plants and even other herbivores in the network. These ef-
fects can be mediated solely by the richness of herbivore species
that interact with a given plant and plant stratum (binary/qualitative
network) or by the combined richness and abundance of herbivores
(weighted/quantitative network). We found that the influence of
PD dimension (structural or compositional) and PD scale (among or
within plants) on network metrics differed for the binary (herbivore
identity) and weighted (herbivore identity and abundance) networks
(Figure 4; Tables S4 and S5).
COSMO et al. Journal of Ecology |
      2481

F I G U R E 2   Variation among seasons and strata of different heights for (a) caterpillar abundance and (b) herbivory (proportion leaf
area lost). The solid line represents the median, the box the 75% quantiles, and the whiskers represents the 95% quantiles. For each
season, data from both sampling dates (July 2017 and April 2018 for the dry season, and October 2017 and January 2018 for the rainy
season) were pooled, representing 62 plants (62 measurements from each height stratum) for each season

F I G U R E 3   Proportion of the total variation that occurred within individual plants for caterpillar abundance, species richness and Hill's
number of the Shannon–­Wiener index (SW index), and the structural and compositional dimensions of phytochemical diversity (PD).
Comp-­PC1 and comp-­PC2 refer to the scores of the two principal components that explained most variation (78.7%) in the compositional
dimension of PD (see Table S1). High values on these principal components represent plants with high concentrations of compounds 1, 2,
10, 12, 13 and 14 (high positive loadings; Table S1) and low concentrations of compounds 3, 4, 5, 6, 7, 8, 9, 11 and 16, some of which were
tentatively identified as Piper amides (high negative loadings; Table S1). Points correspond to mean values, and lines correspond to 95%
confidence intervals estimated via 1,000 bootstraps

In the binary network, the structural dimension at the scale of
the individual plant was the predominant driver and significantly
decreased the capacity of a plant to directly and indirectly affect
the entire network (Figure 4b; Table S4). Both degree (direct inter-
actions) and Katz centrality (direct and indirect interactions through
pathways of all lengths) significantly decreased with increasing plant
structural dimension (β = −0.13 and −4.31 for degree and Katz cen-
trality respectively), while closeness centrality (direct and indirect
interactions through shorter and efficient paths) significantly de-
creased both with increasing the among-­plant structural dimension
(β = −3.24) and increasing comp-­PC1 (β = −2.98) of the compositional
dimension. Plants with low scores on comp-­PC1 were associated
with higher relative amounts of compounds tentatively identified as
Piper amides (Table S1). Therefore, individual plants with low struc-
tural PD and higher relative amounts of amides have a greater direct
and indirect influence in the network through herbivore identity.
 |
2482      
Journal of Ecology COSMO et al.

F I G U R E 4   Representation of the (a) binary plant–­herbivore network and (b) weighted strata–­herbivore network. Red lettered squares
represent caterpillar species. Numbered green circles represent plants in the binary network because only among-­plant metrics were
significant (see panel c) and they represent strata in the weighted network. Green circles with repeated numbers in panel (b) represent
different strata from the same plant. Darker green colours indicate higher levels of the structural dimension of phytochemical diversity
(PD) at the scale of individual plants (a) or strata within plants (b). (c) Coefficient estimates for the mixed models fitted with the binary
(in yellow) and weighted (in blue) versions of degree (direct interactions), closeness (direct and indirect interactions through the shortest
paths) and Katz centrality (direct and indirect interactions through pathways of all lengths) as response variables. Lines represent 95%
confidence intervals. For simplicity, only coefficients of non-­control variables are shown. Comp-­PC1 and comp-­PC2 refer to the scores of
the two principal components that explained most variation (78.7%) in the compositional dimension of PD. High values on these principal
components represent plants with high concentrations of compounds 1,2, 10, 12, 13 and 14 (high positive loadings; Table S1) and low
concentrations of compounds 3, 4, 5, 6, 7, 8, 9, 11 and 16, some of which were tentatively identified as Piper amides (high negative loadings;
Table S1). Please refer to Table S2 for correspondence among letters and caterpillar morphospecies. Tables S4 and S5 contain full model
details

For the weighted network, in contrast, the compositional dimen-
sion at the within-­plant (stratum) scale was the main driver and sig-
nificantly increased the direct and indirect influence of plant stratum
in the network (Figure 4c; Table S5). Comp-­PC2 of the within-­plant
compositional dimension significantly increased all three metrics
(β = 0.38, 5.63 and 13.31 for degree, closeness and Katz centrality
respectively). On the other hand, comp-­PC1 of the within-­plant com-
positional dimension only increased closeness centrality (β = 6.09),
and the within-­plant structural dimension only significantly in-
creased Katz centrality (β  =  12.13). Strata with higher comp-­PC1
COSMO et al. Journal of Ecology |
      2483

scores are associated with higher concentrations of compounds 10,
12, 13 and 14 (Table S1) while strata with higher comp-­PC2 scores
are associated with higher concentrations of compounds 1 and 2
(Table  S1), and these strata are therefore more likely to propagate
direct and indirect effects through both herbivore identity and
abundance in the network. Several compounds tentatively identi-
fied as Piper amides (compounds 3, 4 and 8) had negative loadings
on both comp-­PC1 and comp-­PC2, and therefore within-­plant strata
with high concentrations of these compounds are more isolated in
the network and less likely to propagate direct and indirect effects.
The control variables of individual plant height and season
also significantly affected the binary and weighted version of the
network metrics. The full models with all coefficients are available in
the Supporting Information (Tables S4 and S5).
3.3 | Within and among plants, phytochemical
diversity directly and indirectly affects caterpillar
biodiversity and herbivory
Through the structural equation model (SEM), we again found that
the direction of the effect of PD depends on both the dimension
(structural or compositional) and the scale (among or within plants)
of PD (Figure 5). Within plants, caterpillar abundance increased with

F I G U R E 5   Results for the caterpillar biodiversity structural equation model (SEM). For simplicity, only causal and significant (p < 0.05)
paths of non-­control variables are shown (see Table S7 for all model coefficients). Abundance and species richness refer to caterpillars,
and SW index refers to caterpillar Shannon–­Wiener diversity index. Comp-­PC1 and comp-­PC2 refer to the scores of the two principal
components that explained most variation (78.7%) in the compositional dimension of PD. High values on these principal components
represent plants with high concentrations of compounds 1,2, 10, 12, 13 and 14 (high positive loadings; Table S1) and low concentrations of
compounds 3, 4, 5, 6, 7, 8, 9, 11 and 16, some of which were tentatively identified as Piper amides (high negative loadings; Table S1). Red
arrows denote negative effects, while green arrows positive effects. Coefficients are standardized estimates, except for the effects of
abundance on species richness, and the effect of species richness on species diversity (see Table S6 for full SEM results)
 |
2484      
Journal of Ecology
comp-­PC1 (β = 0.19) and comp-­PC2 (β = 0.12). This indicates that
higher amounts of Piper amides were associated with lower caterpil-
lar abundance, since these compounds had large negative loadings on
both PCs (Table S1). Caterpillar abundance among plants decreased
with increasing structural PD (β  =  −0.27). Caterpillar richness and
caterpillar SW index were not directly affected by PD, but richness
increased with abundance (β = 0.18) and richness positively affected
the SW index (β = 1.27). Therefore, caterpillar richness and SW index
were positively affected (indirectly) by within-­plant compositional
PD, through its positive effect on abundance. Caterpillar richness
and SW index were negatively affected (indirectly) by among-­plant
structural PD, through its negative effect on abundance. Herbivory,
on the other hand, decreases only with the structural dimension of
PD, both within (β = −0.16) and among (β = −0.20) plants (Figure S3).
At the among-­plant scale, the structural dimension of PD decreased
with individual plant height (β = −0.17). However, within plants, struc-
tural PD increased when going from low to high strata (β  =  0.39;
Figure  S2). For the compositional dimension, comp-­PC2 decreased
when going from low to high strata within plants (β = −0.17), indicating
higher strata had higher amounts of Piper amides (Table S2; Figure S2).
Among plants, comp-­PC1 decreased with individual plant height
(β = −0.23), indicating that lower plants had higher amounts of Piper
amides. The structural dimension only weakly negatively correlates
with comp-­PC2 both within and among plants, and weakly positively
correlates with comp-­PC1 only among plants (Table S6; Figure S4).
All variables were affected by season, except for the structural
and compositional dimensions of PD within plants and herbivory
(Table S6). Furthermore, caterpillar abundance increased with plant
leaf number and individual plant height, while herbivory decreased
when going from low to high strata within plants (Table S6).
Sensitivity analysis showed that these SEM results holds for the
entire caterpillar community rather than for only the most abundant
or rare species (Tables S7 and S8).
4 |  D I S CU S S I O N
At what levels of organization do the multiple dimensions of PD
shape insect–­plant interactions? Previously, these interactions
have been shown to be affected at the plant species and individual
levels by both the structural and compositional dimensions of PD
(Glassmire et al., 2016; Poelman et al., 2009; Richards et al., 2015).
However, we still know little about how these dimensions vary
within plants and the consequences of this variation for plant–­insect
interactions when compared to variation among plants. Here we
used the tropical shrub P. amalago and its associated caterpillars as
a model system to disentangle how distinct dimensions of PD drive
insect–­plant interactions across the among-­ and within-­plant scales
of biological organization. Taken together, our results point to three
major properties of how the structural and compositional dimen-
sions of PD shape insect–­plant interactions among and within plants.
First, we show that each dimension of PD can mediate different
ecological processes within individual plants. While the structural
COSMO et al.
dimension decreased herbivore feeding damage, the compositional
dimension increased herbivore biodiversity. In our analyses, the
compositional dimension is composed of two principal components,
and both were negatively correlated with compounds tentatively
identified as P. amalago amides, but positively correlated with other
unidentified compounds. Therefore, within plants it is the structural
diversity of the entire mixture of compounds that regulates the
amount of damage caused by herbivores, while blends rich in Piper
amides indirectly resulted in lower caterpillar richness and diversity.
These findings suggest that the identity of herbivores on a plant
may result from the concentration of specific compounds through
identification of suitable plants and plant tissues (Singer, 2016), but
the structural diversity of compounds in the plant tissue can limit
herbivore feeding damage. These results provide an important link
between the different dimensions of PD and herbivore biodiversity
and highlight that both blend composition and structural diversity
play important roles in structuring plant–­insect communities. It is
important to note, however, that our field study did not allow us to
be certain that the sampling location was the result of a consistent
choice by caterpillars. Furthermore, our design does not allow us
to disentangle constitutive from induced chemical defences, which
could result in a high herbivore biodiversity causing a high PD and not
the other way around. Nevertheless, because herbivore feeding be-
haviour and constitutive/inducible chemical diversity of plant tissues
are heritable traits, our results reinforce previous results showing
that plant–­herbivore co-­evolution can be mediated by the variability
of phytochemicals within plants (Broekgaarden et al., 2010; Utsumi
et al., 2011). For instance, when herbivores feed or oviposit on dif-
ferent plant parts and they are unable to distinguish less defended
plant parts, they can have longer development times and exposure
to predators, lower pupal mass and reduced offspring fitness. These
fitness costs can select for herbivores to discriminate and selectively
feed on less toxic plant parts. Likewise, herbivores that can detect
less defended plant parts can select for unpredictability and thus,
heterogeneity in plant chemical defences (Herrera, 2009, 2017).
Second, the effects of PD at the scale of the individual plant and
the strata within plants have the potential to cascade to the entire
plant population and herbivore community through direct and indi-
rect effects in the plant–­herbivore network. We found that strata
with higher concentrations of specific compounds (high scores on
comp-­PC1 and comp-­PC2, Table S1), but from individual plants with
low structural PD, were more likely to directly affect the herbivores
feeding upon them, indirectly affect all other herbivores in the com-
munity and indirectly affect all the other plants in the population
(Figure  4). Hence, our results provide evidence that the composi-
tional and structural dimensions of PD can have distinct effects on
network structure. Furthermore the scale (individual or plant part)
at which each of these dimensions varies influences the network.
Recent theory shows that in networks, nodes that can propagate
more direct and indirect effects can drive stronger and more pre-
dictable extinction cascades (Pires et al., 2020). Our results, there-
fore, suggest that variation in chemical traits both among and within
plants is key to understanding and predicting how disturbances and
COSMO et al.
extinctions affect the associated herbivore community and entire
plant populations. Another extension of these results, together with
our results that the structural dimension of PD decreases herbivore
feeding damage, is the possibility that managing PD in agroeco-
systems could be essential for limiting pest damage. For instance,
it could be highly beneficial to manage agroecosystem diversity to
increase overall structural PD throughout the agroecosystem, thus
reducing damage to crops. It is quite likely that many as yet unknown
mechanisms related to PD are involved in the well-­known benefits
of agroecosystem diversification for agroecosystem services and
sustainable crop production, and we believe this area is a promis-
ing one for future research (Hauri et al., In Press; Silva et al., 2018;
Vandermeer et al., 2019).
Third, PD may be maintained by how its multiple dimensions
interact with herbivores and mediate ecological and evolutionary
processes on different scales of biological organization. Our results
show that the structural dimension varies more among plants than
within plants. However, within plants, even a small amount of varia-
tion in this dimension was enough to decrease herbivory. In contrast,
the compositional dimension varied more and affected herbivore
abundance only within plants, while herbivore abundance among
plants decreased only with the structural dimension (Figures 3 and
5). This scale dependence of the effects of PD may be mediated by
how different herbivores respond to variation of the dimensions of
PD at different scales. Herbivores differ in the scales at which they
make feeding decisions, from the plant part, plant individual to the
plant patch, and PD is known to have different effects on specialists
and generalists (Emerson et  al.,  2012; Massad et  al.,  2017; Moore
et al., 2010). In turn, this interaction of PD between scales is likely
to be a major driver of the evolution of local and systemic induced
responses, through the maximization of chemical noise, as proposed
by Kessler and Kalske (2018).
Our results show that the structural and compositional dimen-
sions of PD may vary and shape insect–­plant interactions on dif-
ferent scales. The question then emerges of how these processes
transfer from the finer within-­plant scale to the broader among
individual and species scales. Or, paraphrasing Levin (1992), how
do we scale from leaf chemistry to the plant–­herbivore community
to the landscape and beyond? A major development in this direc-
tion was the proposal of the concept of a phytochemical landscape
(Hunter, 2016). In this context, we conclude that for herbivorous in-
sects, even a single individual host plant may represent an entire,
fine-­grained phytochemical landscape (Herrera,  2009). Thus, we
expand upon Hunter's (2016) hypothesis and propose that the phy-
tochemical landscape emerges as different layers across scales of
biological organization, within which the different dimensions of PD
can be the dominant components and have distinct scale-­dependent
effects. How information perceived by herbivorous insects is trans-
ferred across these layers remains an open and exciting question.
Therefore, we argue that to indeed scale from the leaf to the ecosys-
tem, future studies should focus on disentangling how the dimen-
sions of PD and herbivore responses are connected across multiple
scales and synergistically drive insect–­plant interactions.
Journal of Ecology |
      2485
AC K N OW L E D G E M E N T S
The authors are grateful to the Fundação Serra do Japi, in par-
ticular to Vânia P. Nunes, for logistical support and for allowing
them to carry out their study at the Reserva Biológica Municipal
da Serra do Japi. They thank Paulo R. Guimarães Jr, Débora C.
Rother, André V.L. Freitas, Mariana A. Stanton, Tara J. Massad and
Mathias M. Pires for discussions and suggestions during planning,
data analysis and manuscript writing. They are grateful to Luiz H.
Rezende, João V. Suzigan and Juliana Rink for help with the field
work. They thank two anonymous reviewers whose comments
greatly improved the manuscript, and Tobias Züst for help during
the submission process. This study was financed by a FAPESP-­NSF
Dimensions of Biodiversity-­Biota collaborative grant ‘Chemically
mediated multi-­t rophic interaction diversity across tropical gra-
dients’ (2014/50316-­7 ) and by CAPES-­F inance Code 001. M.J.K.
has financial support from CNPq (304607/2019-­3). M.P. received
support from FAEPEX-­UNICAMP (grant no 60412 and PAPDIC
2014/4715). R.C. is funded by FAPESP (2013/25991-­0), CNPq
(307015/2015-­7 and 307447/2018-­9) and a Newton Advanced
Fellowship from the Royal Society. L.G.C. currently receives a
FAPESP scholarship (2019/22146-­3). The authors declare no con-
flict of interests.
AU T H O R S ' C O N T R I B U T I O N S
L.G.C., M.P. and R.C. designed the study; L.G.C. and M.P. collected
the field data; L.G.C., L.F.Y. and M.J.K. performed chemical analysis;
L.G.C., G.M.F.F., M.P. and R.C. conducted statistical analyses; L.G.C.
wrote the first draft of the manuscript. All the authors contributed
substantially to further revisions.
DATA AVA I L A B I L I T Y S TAT E M E N T
Data available from the Dryad Digital Repository https://doi.org/​
10.5061/dryad.p8cz8​w9mz (Cosmo et al., 2021).
ORCID
Leandro G. Cosmo  https://orcid.org/0000-0003-2541-2645
Lydia F. Yamaguchi  https://orcid.org/0000-0003-2305-8208
Gabriel M. F. Felix  https://orcid.org/0000-0002-3901-4333
Massuo J. Kato  https://orcid.org/0000-0002-3315-2129
Rodrigo Cogni  https://orcid.org/0000-0001-9907-9297
Martín Pareja  https://orcid.org/0000-0002-2615-9947
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Cosmo LG, Yamaguchi LF, Felix GMF,
Kato MJ, Cogni R, Pareja M. From the leaf to the community:
Distinct dimensions of phytochemical diversity shape
insect–­plant interactions within and among individual plants.
J Ecol. 2021;109:2475–­2487. https://doi.
org/10.1111/1365-­2745.​13659