263

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 263-276, January-December 2022 (Published Apr. 30, 2022)

Which variables influence the herbivory amount

on Montrichardia spp. (Araceae) in aquatic ecosystems?

Ana Luisa Biondi Fares1; https://orcid.org/0000-0002-2738-1670

Wendell Vilhena de Carvalho2; https://orcid.org/0000-0002-3494-7708

Thaisa Sala Michelan1*; https://orcid.org/0000-0001-9416-0758

Grazielle Sales Teodoro1; https://orcid.org/0000-0002-5528-8828

1. Laboratório de Ecologia de Produtores Primários, Instituto de Ciências Biológicas, Universidade Federal do Pará,

Belém, Pará, Brazil; afaresbiondilima@gmail.com, tsmichelan@ufpa.br (Correspondence*), gsales.bio@gmail.com

2. Museu Paraense Emilio Goeldi, Belém, Pará, Brazil; wendell_vilhena@hotmail.com

Received 13-VIII-2021. Corrected 21-II-2022. Accepted 21-IV-2022.

ABSTRACT

Introduction: The study of herbivory is fundamental in ecology and includes how plants invest in strategies and

mechanisms to reduce herbivore damage. However, there is still a lack of information about how the environ-

ment, plant density, and functional traits influence herbivory in aquatic ecosystems.

Objective: To assess if there is a relationship between herbivory, environmental variables, and plant traits two

species of Montrichardia, a neotropical aquatic plant.

Methods: In September 2018, we studied 78 specimens of Montrichardia arborescens and 18 of Montrichardia

linifera, in 18 sites in Melgaço, Pará, Brazil. On each site, we measured water depth, distance to the margin, and

plant density. From plants, we measured plant height and leaf thickness, and photographed the leaves to calculate

the specific leaf area and percentage herbivory. To identify anatomical structures, we collected fully expanded

leaves from three individuals per quadrat.

Results: For M. arborescens, plants with thicker leaves and higher specific leaf area have less herbivore dam-

age. For M. linifera, plants from deeper sites and with thicker leaves had more herbivore damage, while plants

that grew farther from the margin had less damage. We found anatomical structures related to defense, such as

idioblast cells with phenolic compounds, and cells with solid inclusions that can contribute to avoiding severe

damage.

Conclusions: Herbivory in these Montrichardia species can be explained by a combination of plant and envi-

ronmental traits (patch isolation and water depth). The main plant traits are leaf thickness and area, but chemical

compounds and solid inclusions also help Montrichardia to sustain less damage than other macrophytes.

Key words: aquatic plants; functional traits; chemical defense; plant-herbivore interactions; freshwater ecosys-

tems; Amazon.

https://doi.org/10.15517/rev.biol.trop..v70i1.48076

AQUATIC ECOLOGY

Antagonistic biological relationships, such

as herbivory, are fundamental to understand

plant-animal relationships, coevolution, and

how plants can invest in different strategies to

avoid consumption by animals, involving dif-

ferent defense mechanisms (Poore et al., 2012;

Turcotte et al., 2014). Herbivory differs among

terrestrial and aquatic ecosystems, being higher

in the latter (Bakker et al., 2016; Cebrian &

Lartigue, 2004; Reese et al., 2016). Herbivores

remove on average 40-48 % of plant biomass

in freshwater and marine ecosystems, which

264 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 263-276, January-December 2022 (Published apr. 30, 2022)

is typically 5-10 times greater than what is

reported in their terrestrial counterparts (Bak-

ker et al., 2016). In addition, herbivores affect

aquatic macrophytes abundance and species

composition and therefore alter the function-

ing of aquatic ecosystems, including primary

production, biogeochemical cycling, and car-

bon stocks (Bakker et al., 2016; Chaichana

et al., 2011). However, as much as there is

a substantial literature assessing the factors

influencing herbivory on macrophytes and the

consequence of it on the ecosystem (see Bak-

ker et al., 2016 for a synthesis on the subject),

there is still a lack of information regarding the

variables (environmental, community charac-

teristics and/or plant functional traits) that can

influence herbivory in neotropical aquatic eco-

systems and how it may contribute to the high

herbivory in these habitats.

Factors related to plant community struc-

ture, such as plant density and diversity, are

generally used as measurement of resource

quantity and may influence herbivore abun-

dance and, consequently, the damage in plants

(Kim & Underwood, 2015; Lacey et al., 2014;

Otway et al., 2005; Root, 1973). There is not

a clear pattern related to plant density on the

herbivory amount, once they can increase the

damage through changes in herbivory loads,

known as resource concentration effect hypoth-

esis (Kim & Underwood, 2015; Root, 1973)

or decrease the amount of herbivory, referred

as the dilution effect hypothesis (Otway et al.,

2005; Wenninger et al., 2016). So far, these

hypotheses have not been tested on the aquatic-

terrestrial interface. Furthermore, in aquatic

ecosystems, factors such as water depth and

distance of macrophytes banks to the margin,

as an isolation patch measurement, also may

influence plant exposition and visibility to

herbivores (Kolb, 2008; Korpinen et al., 2007).

In this context, plants have evolved a

wide array of defense traits that provide pro-

tection against herbivory attacks, including

physical traits (e.g.: presence of trichomes,

spines, height, leaf toughness) and chemi-

cal compounds (e.g.: secondary metabolites,

solid inclusions, hormonal responses, ergastic

substances) (Agrawal & Fishbein, 2006; Paro-

lin, 2009; Poore et al., 2012). Among physical

traits, plant height and size are often related

to competition and dispersal ability and are

linked to environmental conditions (Schlinkert

et al., 2015). However, those traits also make

plants more susceptible to herbivory as those

individuals become bigger and more exposed

to the associated fauna, thus providing more

microhabitats and food resources for them

(Díaz & Cabido, 2001; Shlinkert et al., 2015).

Leaf traits are also associated to herbivory

(Poorter et al., 2009; Reese et al., 2016). Leaf

thickness is correlated with leaf toughness, a

trait negatively associated to herbivory. Gener-

ally, thinner leaves are considered more palat-

able than thicker leaves (Guerra et al., 2010),

because they are less tough, have less lignin

and other compounds that toughen them, thus

becoming easier to be consumed by herbi-

vores (Smilanich et al., 2016). Furthermore,

leaves with lower specific leaf area (SLA)

are considered more resistant to herbivory,

being generally avoided (Pérez-Harguindeguy

et al., 2013; Poorter et al., 2009; Reese et al.,

2016). SLA varies closely with other traits that

can be related to herbivory defense, it can be

positively related to mass-based leaf nitrogen

content (which has more nutrient content,

increasing the preference of herbivores), and

negatively related to leaf longevity (higher leaf

lifespan generally shows higher carbon invest-

ment, making leaves less palatable and less

preferred by herbivores) (Pérez-Harguindeguy

et al., 2013; Wright et al., 2004). Furthermore,

regarding the defense system against herbi-

vores, plants can produce a pool of toxic, deter-

rent, and volatile compounds (Bari & Jones,

2008; Gross & Bakker, 2012), and this reflects

directly in plant physiology and in cell anatomy

(Zunjarrao et al., 2019).

Thus, our aims were to assess if there is

a relationship among herbivory amount and

environmental variables (e.g.: water depth and

distance of the margin), plant density, and plant

structural traits (e.g.: height, leaf thickness,

and SLA) in two Montrichardia (Araceae)

species, being M. linifera and M. arborescens.

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We aimed to answer the following question:

how environmental factors, plant density and

plant traits influence plant herbivory? Our

hypotheses were: i) considering the environ-

mental factor, the herbivory amount is higher

in plants occurring in deep places that gener-

ally are more distant from the margin, thus

more visible; ii) concerning plant density, we

expected lower herbivory in sites with dense

patches, once they have more resource quantity

(dilution effect); iii) regarding plant traits, we

expected higher herbivory in taller plants, with

thinner leaves and/or higher SLA as they are

less scleromorphic and therefore, more palat-

able. In addition, we also aimed to indicate if

there are, in both species, anatomic structures

acting together with plant structural traits in the

plant’s defense system, like ergastic substances

and solid inclusions.

MATERIALS AND METHODS

Study area, sampling design and spe-

cies: Sampling took place across the Curuá

River, inside the Caxiuanã National Forest

(FLONA-Caxiuanã), located in the municipal-

ity of Melgaço, Pará, Brazil (1°47’32.3” S &

51°26’2.5” W) near the Ferreira Penna Scien-

tific Station, in September 2018. This side of

the FLONA is characterized for having black

water rivers that form a type of forest called

Igapós, and for the presence of various macro-

phyte banks and islands.

In total, we sampled 18 sites that had Mon-

trichardia species, and a total of 96 individuals,

being 78 Montrichardia arborecens specimens,

and 18 M. linifera specimens. We used a motor-

ized boat to get to the stands where we saw

the species and maintained at least 60 m of

distance between surveyed locations.

Plant description: The Montrichardia

Crueg (Araceae) genus consists of two spe-

cies (Montrichardia linifera (Arruda) Schott

and Montrichardia arborescens (L.) Schott)

of large emergent plants widely distributed

in the Neotropics (Croat et al., 2005), with a

wide distribution in the Amazonian ﬂoodplain

(Gibernau et al., 2003; Lopes et al., 2016).

Individuals of Montrichardia can grow up to 6

meters in height and the populations (alone or

together with other species) form rooted stands,

called matupás (De Freitas et al., 2015) of vary-

ing sizes, from a few meters to large ﬂoating

islands, occurring in different distances from

the margin (Lopes et al., 2016). These float-

ing islands appear with the release of a part of

the macrophytes bank from the margins, the

plants and sediment adhered to the roots form

these islands can move in the rivers according

to the water flow. Although these plants do

not seem to be the most preferred hosts for

some herbivores and parasites (as plant extracts

show antibacterial, cytotoxic and insecticide

activities), especially because of the defense

mechanisms present on these organisms (e.g.

chemical defense, with cells that produce alka-

loids and other secondary metabolites) (San-

tos et al., 2014), the literature suggests these

plants’ leaves and fruits are food source for

fish, turtles, and big mammal such as manatees

and cattle (Amarante et al., 2010). Montrich-

ardia spp. present high phenotypic plastic-

ity in response to the environment (Lopes et

al., 2016) and participate in processes on the

aquatic-terrestrial ecotone, such as stabilizing

river margins (Amarante et al., 2011).

Both species of Montrichardia are fre-

quently recorded along rivers and lakes in the

Amazon basin. Montrichardia linifera is more

frequently recorded along riverbanks and M.

arborescens occupies a position along the

floodplain and the fringe in the understory of

ﬂoodplain forests (Lopes et al., 2016). The

species can occur in both nutrient-rich envi-

ronments, such as white-water rivers, and in

nutrient-poor environments, such as the black-

water Rivers, and brackish environments in

the estuary of the Amazon River (Lopes et al.,

2016). These species play an important role in

the stabilization of river banks in the Amazon

floodplains (Macedo et al., 2005).

Environmental and ecological measure-

ments: To test the effect of environmental and

ecological variables on the herbivory amount of

266 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 263-276, January-December 2022 (Published apr. 30, 2022)

Montrichardia we used 18 samples measured

using a 1 m2 (1 x 1 m) quadrat which was

placed in areas we had access to collect the

species within each sampling site. In each 1 m2

quadrat we measured two environmental char-

acteristics, i) water depth, using a graduated

stick (cm) and ii) distance estimated from the

plot to the riverbank using tape measure (m).

Additionally, related to ecological/community

factors we measured plant density, by visually

assessing the percentage of cover (%) of Mon-

trichardia species inside the 1 m2 quadrat.

Plants measurements: In each quadrat,

we measured the height (cm) of each Mon-

trichardia individual, and we selected 2 leaves

of each individual to analyze the leaf traits at

the laboratory. We always chose mature leaves,

that were generally in the middle of the stem,

thus we excluded older (more at the base of the

stem) and younger (more at the apex) leaves,

that could cause a bias in the data.

At the laboratory, we measured the leaf

traits following the protocol proposed by

Pérez-Harguindeguy et al. (2013). We mea-

sured leaf thickness (LT, mm) of each leaf in

three parts (base, middle and apex) with a digi-

tal micrometer (precision of 0.01) and we took

photos of each leaf to estimate leaf area and to

assess the amount of herbivory on each leaf,

using ImageJ, a free software. To obtain the

herbivory amount, we first calculated the total

area of the leaves, then the areas without her-

bivory damage (holes). By subtracting the area

without holes from the total area, we obtained

the true herbivory area on the leaf, which was

then transformed into a percentage value (using

the equation herbivory area (m2) * 100 / total

leaf area (m2)). Posteriorly, the leaves were

oven-dried at 65 °C for 72 hours, and then

weighted using a digital scale (precision 0.01

g), to obtain dry mass values. We calculated

the specific leaf area (SLA), using the equation

SLA = leaf area (m2)/ dry mass (kg).

Anatomical analysis: To qualitatively

identify anatomical structures related to plants

defense, we evaluated anatomical leaves slices.

For this purpose, fully expanded leaves were

collected from three individuals per quadrat

(N = 22), generating a total of 66 samples.

The middle, midrib and apex region of the leaf

blade were fixed in FAA 70 % (70º ethanol,

formaldehyde and 18:1:1 v/v acetic acid), later

washed in running water and dehydrated in

ethylic series with alcohol 70 % for storage and

to absolute ethanol to be processed (Johansen,

1940). The permanent slides were made after

inclusion of the material in paraffin and sec-

tioned transversally in a rotary microtome. The

sections were stained in astra blue and aqueous

safranin at 1 %, after assembled with Canada

Balsam, according to the usual techniques

compiled by Kraus and Arduin (1997). The

anatomical analysis and the photomicrographs

were taken using the photomicroscope Leica

Microscope DMLB, attached to an image cap-

ture system.

The visual analysis through light micros-

copy was qualitative, analyzing the cell struc-

tures and compounds that can be related to

plant defense, such as idioblast cells with

secondary metabolites and cells with solid

inclusion by comparing the structures seen in

the already published papers for the Montrich-

ardia specie and family (Amarante et al., 2015;

Costa et al., 2009; Ferreira et al., 2006; Macedo

et al., 2005).

Data Analyses: Prior to all hypothesis-

testing analyses, we performed a Pearson Cor-

relation test with all predictive variables, to

assess for multicollinearity. No variable pre-

sented collinearity (correlations were R ≤ 0.6),

so all were included on the next analyses. To

test if the amount of herbivory is higher in

more distant, less dense and deep patches, and

in taller plants with thinner leaves and higher

SLA we performed a binomial Generalized

Linear Mixed Model (GLMM), for each spe-

cies separately. GLMMs are fitted for data with

non-normal distributions and can incorporate

random effects to account for nested observa-

tions (Bolker et al., 2009; Zuur et al., 2009).

For this analysis, we only used as samples

individuals that were found in monospecific

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stands for each species, totaling 78 samples

for M. arborescens and 18 for M. linifera. We

included as predictors the environmental vari-

ables (water depth and distance to the margin),

plant density, and plant traits (plant height,

leaf thickness and SLA), and the amount of

herbivory as the response variable. Since our

response variable was a measure of proportion,

we used the Binomial distribution family on the

model (Zuur et al., 2009). At first, we build a

Generalized Linear Model (GLM) using all the

predictors, to perform a model selection (using

Forward stepwise) based on the lowest Akaike

Information Criterion (AIC) score, and thus

select the best model explaining the variation in

the data. After that, we built a GLMM with the

fixed variables that were selected and includ-

ing the plot as random effects in the model,

because these factors were not part of the

hypotheses but we felt could affect the results.

All continuous variables were standardized.

This final model was validated by checking the

residuals of the analysis (Zuur et al., 2009).

We used the software R (R Core Team,

2020) to perform all analyses. For the model

selection, we used the function ‘stepAIC’ in the

MASS package (Venables & Ripley, 2002). We

used the function ‘glmer’ in the lme4 package

(Bates et al., 2015) to perform the GLMM.

RESULTS

For Montrichardia arborescens, the dens-

est site had 60 % of cover, while the lowest,

15 % (Mean: 28.269 ± 12.889 SD). The tallest

individual was 3.40 m high, while the shortest,

0.90 m high (Mean: 130.380 ± 0.568). For the

plant measurements, the highest leaf thickness

value was 0.4 mm, and the lowest, 0.1 mm

(Mean: 0.271 ± 0.063) and the highest SLA

value was 28.004 m2 kg-1, while the lowest,

10.07 m2 kg-1 (Mean: 15.194 ± 3.787). The

highest herbivory amount in an individual was

23.90 %, while the lowest was 0 (no herbivory)

(Mean: 4.962 ± 5.724). For the environmental

variables, the highest water depth in a plot

was 155 cm while the lowest, 33 cm (Mean:

79.205 ± 31.662) and the highest distance from

the margin was 50 m, while the lowest was 3

m (Mean: 13.846 ± 10.628). Furthermore, for

plant density of M. linifera, the densest site

had 60 % of Montrichardia cover, while the

lowest, 20 % (Mean: 38.333 ± 2.832). The

highest individual was 3.15 m high, while the

shortest was 1.35 m high (Mean: 40.160 ±

0.471). The highest leaf thickness value was

0.4 mm, and the lowest, 0.3 mm (Mean: 0.344

± 0.048) and the highest SLA value was 21.99

m2 kg-1, while the lowest, 8.87 m2.kg-1 (Mean:

11.962 ± 2.832). The highest herbivory amount

in an individual was 10.01 %, while the low-

est was 0.61 % (Mean: 2.581 ± 2.655). For

the environmental variables, the highest water

depth in a plot was 140 cm while the lowest, 83

cm (Mean: 103.389 ± 20.024) and the highest

distance from the margin was 25 m, while the

lowest was 10 m (Mean: 16.833 ± 4.176).

According to the model selection, the best

model to explain the amount of herbivory on

M. arborescens had the variables plant den-

sity, leaf thickness, water depth and SLA (AIC

= 823.5). The GLMM results showed that,

among those variables, the amount of herbivory

was associated negatively with leaf thickness

(Estimate = -2.866, P = 0.007, Fig. 1A) and

SLA (Estimate = 0.082; P < 0.001, Fig. 1B)

(Table 1). For M. linifera, the selected model

had the variables plant density, leaf thickness,

water depth and distance to the margin (AIC =

100.2). The results showed that, among those

variables, the amount of herbivory was posi-

tively associated with leaf thickness (Estimate

= 15.964, P < 0.001, Fig. 2A) and water depth

(Estimate = 0.119, P < 0.001, Fig. 2B), and

associated negatively with the distance to the

margin (Estimate = -0.543, P < 0.001, Fig. 2C).

In relation to leaf anatomy (for both spe-

cies), leaves were dorsiventral with stomata

present only on the abaxial epidermis (Fig.

3). The epidermal contended a single cell

layer with thick anticline and periclinal walls

covered with an evident cuticle (Fig. 3A).

We found anatomic and chemical structures

related to defense, such as the idioblast cells

with phenolic compounds and cells with solid

268 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 263-276, January-December 2022 (Published apr. 30, 2022)

Fig. 1. Plot of the significant effects of the Generalized Linear Mixed Model performed for M. arborescens in the Caxiuanã

National Forest, Brazil. A. Effect of leaf thickness (mm) on leaf herbivory amount of Montrichardia arborescens; B. Effect

of specific leaf area on leaf herbivory amount of Montrichardia arborescens.

TABLE 1

Result of the Generalized Linear Mixed Models performed with variables selected by forward stepwise

evaluating the relationship between herbivory in Montrichardia species and explanatory variables,

in the Caxiuanã National Forest, Brazil

Montrichardia arborescens Montrichardia linifera

Estimate Std. Error z P Estimate Std. Error z P

Intercept -0.669 0.695 -0.962 0.336 Intercept -12.438 2.582 -4.816 < 0.001

Plant Density -0.018 0.016 -1.151 0.250 Plant Density -0.007 0.024 -0.290 0.772

Leaf Thickness -2.866 1.069 -2.682 0.007 Leaf Thickness 15.964 2.499 6.388 < 0.001

Water Depth -0.001 0.006 -0.194 0.846 Water Depth 0.119 0.036 3.346 0.001

SLA -0.082 0.017 -4.725 < 0.001 Distance to the margin -0.543 0.141 -3.847 < 0.001

Random Effects Random Effetcs

Variable Variance Std. Dev. Variable Variance Std. Dev.

Plot 0.441 0.664   Plot < 0.001 < 0.001  

Values in bold indicate significant relationships.

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inclusions - druses, like calcium oxalate crys-

tals (Fig. 3B, Fig. 3C, Fig. 3D, Fig. 3E).

Solid inclusions were observed both in

mesophyll and in midrib parenchimal cells. A

high number of druses were found inside idio-

blasts in the leaf apex, both in the palisade and

spongy layers. The idioblast cells with phenolic

compounds were found in midrib (Fig. 3F).

The phenolic compounds were present in leaf

in the palisade and spongy tissues, also along

the cortical and parenchimal region in the

midrib (Fig. 3F). The phenolic idioblasts have

a varied shape, occupying a large part of the

tissues (rounded to elongated) (Fig. 3G).

DISCUSSION

According to our results, our hypothesis

was partially corroborated, in which the envi-

ronmental factors and plant traits explained the

observed herbivory amount in Montrichardia

sp. Contrary that we predicted, the plant den-

sity was not related to the herbivory amount

in the studied species. For M. arborescens the

leaf traits, such as leaf thickness and SLA, were

related to the herbivory amount. While in M.

linifera both leaf traits, such as leaf thickness,

and environmental factors, as water depth and

distance to the margin, were related to the her-

bivory amount.

Fig. 2. Plot of the significant effects of the Generalized Linear Mixed Model performed for M. linifera in the Caxiuanã

National Forest, Brazil. A. Effect of leaf thickness (mm) on leaf herbivory amount of Montrichardia linifera; B. Effect of

water depth on leaf herbivory amount of Montrichardia linifera; C. Effect of the distance to the margin on leaf herbivory

amount of Montrichardia linifera.

270 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 263-276, January-December 2022 (Published apr. 30, 2022)

Among plant traits that were measured, in

both species leaf thickness was related to her-

bivory amount, however in M. arborescens it

was associated negatively and in M. linifera the

proportion of herbivory was positively associ-

ated with leaf thickness. The negative relation-

ship that we observed in M. arborescens can be

associated with the fact that leaf thickness is

related to structural defense, in which thicker

leaves tend to have greater structural resistance

against leaf-chewing herbivores (Onoda et al.,

2011). Thus, plants can produce thicker leaves

to defend themselves against their herbivores.

Muiruri et al. (2019), found that leaf thick-

ness had a significant effect on herbivory load,

reducing gall abundance in thicker leaves.

Furthermore, they showed that physical traits

might be more important determinants of plant

herbivory than nutritive and chemical defense

traits. Additionally, plant traits, such as the

leaf thickness, can affect the transmission of

vibrational signals and cues that can be used

by animals to plant detectability. Insects, as the

caterpillars, can use plant-borne vibrations to

extract and use information from their biotic

and abiotic environment (Velilla et al., 2020),

influencing in the herbivory load. For example,

plants can evolve thicker leaves to avoid herbi-

vores, however, they may reduce effective use

of vibratory cues by the predators and parasites

that can eat these herbivores (Velilla et al.,

2020). This can be one hypothesis to explain

Fig. 3. Cross section of Montrichardia spp. leaves: A. leaf blade – M. linifera; B. leaf apex - M. linifera; C. phenolic

idioblasts - M. arborescens; D. druse - M. linifera; E. druse under polarized light - M. linifera; F. midrib - M. arborescens; G.

leaf blade - M. arborescens. Dr: druse, Pc: phenolic compounds, St: stomata, Eab: abaxial epidermis, Ead: adaxial epidermis.

Bars: C. D. and E. = 15 µm, A. B. and G. = 50 µm, F. = 100 µm.

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a positive relation between herbivory load and

leaf thickness, as we observed in M. linifera.

We were not able to identify the herbivore

species that were damaging Montrichardia

individuals, but on more than one occasion

we saw caterpillars and eggs on the leaves

(personal observation).

The SLA was also negatively related to

the herbivory load in M. arborescens. SLA is

generally positively related to plant growth rate

and leaf quality (Milla et al., 2008), in which

low SLA values indicate a high investment in

carbon to build leaves and higher leaf longevity

(Wrightet al.,2004). There are some interspe-

cific herbivory patterns in diverse land commu-

nities related to functional traits, in which, in

some communities, leaves with lower SLA are

less eaten by herbivores (Poorter et al., 2009;

Reese et al., 2016). However, we observed a

negative relationship between SLA and her-

bivory, in which leaves with higher carbon

investments were more eaten. This possibly can

be associated to higher leaf longevity (Pérez-

Harguindeguy et al., 2013; Wright et al., 2004),

being exposed to herbivores for more time than

leaves with short lifespan.

Insect herbivory patterns are influenced

by multiple ecological drivers acting at differ-

ent spatial scales (Wang et al., 2022). In M.

linifera the herbivory amount was negatively

associated with the bank distance to the river

margin. This patch distance can be viewed as

an isolation measurement, because fragmented

populations are less visible and less apparent to

insect pollinators, moreover, visitors (including

grazers and pollinators) (Kolb, 2008). Isola-

tion is considered an important factor affecting

insect herbivory, in which, insect herbivores

tend to reach higher densities in patches that

can be accessed and colonized more easily

(Wang et al., 2022). The patch isolation can

also decrease insect herbivory via changes in

abiotic factors, such as humidity, wind and

temperature, and resource limitation (i.e. “bot-

tom-up” factors) that affect herbivores directly

(Maguire et al., 2016).

The herbivory amount in M. linifera

was positively associated with water depth.

Resource availability (nutrients, light) declines

exponentially with increasing depth (Bakker et

al., 2016). The water motion and light levels

also declines, reducing photosynthesis and

nutrient uptake rates, making these areas more

vulnerable to being consumed by animals,

increasing grazing efficiency and herbivore

density (Korpinen et al., 2007). It is possible

that the herbivory amount in M. linifera is

increasing because of these factors (although

the range of water depth found in our study is

not that high - 140 cm to 83 cm), which could

indicate that the species that is inhabiting a

more ‘resource limiting environment’ are more

susceptible to herbivore damage.

Among the strategies, some of these mech-

anisms are compartmented inside the plant

(Giordano et al., 2020), like chemical com-

pounds (i.e. secondary metabolites, solid inclu-

sions, hormonal responses, ergastic substances)

(Agrawal & Fishbein, 2006; Carmona et al.,

2011), to avoid being consumed by animals.

As anatomical defense structures, the idioblast

cells with phenolic compounds seen in both

Montrichardia species play a central role in

this group, including acting as a barrier to

several pathogens that could prevent infection

diffusion, penetration of fungi, defense against

various bacterial and damage caused by insects

or by grazing animals, like herbivores (Liang

et al., 2007; Zhang et al., 2019). Phenols are

aromatic compounds derived from the shi-

kimic acid pathway and also protect cells from

UV-B radiation and oxidative stress (Berli et

al., 2010; Uleberg et al., 2012). The phenolic

compounds seen in the idioblasts along the

Montrichardia leaf blade corresponding to

substances in the flavonoid group (Amarante

et al., 2015), these phenolic compounds have

pharmacological and anti-nutritional action,

inhibiting lipid oxidation and fungal prolif-

eration (Soares, 2002). The plant normally uses

these phenolic compounds as an antiseptic and

to protect itself against dehydration, rot and

attack by animals (Ferreira et al., 2006). Our

phenolic structures are similar to those found

by Amarante et al. (2015).

272 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 263-276, January-December 2022 (Published apr. 30, 2022)

Allied to that, solid calcium inclusions are

widespread in mesophyll and in midrib paren-

chymal cells for both Montrichardia species.

Calcium oxalate crystals is the most prominent

storage of calcium salts (Fahn, 1990) and in

leaf blades it protects plants from biotic and

abiotic stresses (Giordano et al., 2020). These

specialized cells are responsible for metal

detoxification (Franceschi & Nakata, 2005),

light scattering (Gal et al., 2012), high-capacity

calcium (Ca) regulation, can sequester heavy

metals ions and mainly protection against

herbivores, for being a multifaceted roughly

spherical structure that is unpalatable to herbi-

vores (Giordano et al., 2020; Pierantoni et al.,

2018). The presence of calcium oxalate crystals

is one of the outstanding characteristics of the

Araceae family (Costa et al., 2009; Ferreira

et al., 2006), seen by Amarante et al. (2015).

One hypothesis considered as the cause of the

toxicity on those plants from these family is the

fact that these crystals, in the form of druses

or raffids, are associated with toxic substances

and are found in the latex expelled by the plant

(Martins et al., 2005). In humans, the latex with

druse content causes burns, rashes and spots

on the skin and in contact with the eyes can

even cause blindness (Amarante et al., 2011;

Macedo et al., 2005).

Thus, these two advantageous structures

of cellular metabolism, idioblasts cells with

phenol and solid inclusions (like druses) on

both Montrichardia species, are all part of the

plant defense system and it is influenced by

various genetic and environmental factors, as

seen in other species from the Araceae fam-

ily (Ferreira et al., 2006) and Montrichardia

species (Amarante et al., 2011; Macedo et al.,

2005). Nutrient availability is a determinant

in the allocation of metabolites for defense

against herbivores (Izzo et al., 2018). Overall,

these strategies are considered anti-herbivore

defenses in freshwater macrophytes and sea-

grassess (Bakker et al., 2016), making leaves

with low palatability to keep pathogens, from

bacteria and fungi up to insects and other herbi-

vores, away (Giordano et al., 2020). This mean

the plant’s defense mechanisms are effective,

probably acting immediately after an attack and

preventing severe damages (induced response),

or only sustaining damage from specialist

herbivores that evolved to overcome those

defenses (Ali & Agrawal, 2012; Mithöfer &

Boland, 2012), or a combination of both (Ali &

Agrawal, 2012).

Concluding, our results indicate that

herbivory in Montrichardia species could be

explained by combination of environmental

(patch isolation and depth water) and plant

traits. We found that leaf traits were important

factors that drive changes in herbivory load,

mainly leaf thickness and specific leaf area.

Furthermore, Montrichardia species invest in

chemical compounds and solid inclusions to

avoid severe damage on the leaves, thus may

sustain less damage than other macrophyte

species. Our findings bring new information

regarding which set of variables explain the

herbivory amount in aquatic macrophytes,

emphasizing the importance of landscape iso-

lation, leaf traits and defense compounds on

those organisms in freshwater ecosystems.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We thank “De Leon” for helping during

field work and the entire team of professors,

students, collaborators and staff in the Field

Ecology Course in Caxiuanã, Emmy F. M.

Espinoza for helping to translate the abstract

in Spanish and three anonymous reviewers

who helped a lot with comments and sug-

gestions to improve this work. We are also

grateful to the Coordenação de Aperfeiçoa-

mento de Pessoal de Nível Superior (CAPES)

for funding the senior internship scholarship

273

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 263-276, January-December 2022 (Published Apr. 30, 2022)

for TSM to conduct research at University

of Florida (PROCAD-AMAZONIA process

88881.465213/2019-01). This work was sup-

ported by “Establishing a Brazilian-Norwegian

master course in tropical rainforest ecology

and biodiversity” project (UTFORSK 2017

number UTF-2017-four-year/10053), CAPES

– Finance Code 001 and by Conselho Nacional

de Desenvolvimento Científico e Tecnológico

CNPq – process 433125/2018-7.

RESUMEN

¿Qué variables influyen en la herbivoría en

Montrichardia spp. (Araceae) en los ecosistemas

acuáticos?

Introducción: La herbivoría es fundamental para com-

prender cómo las plantas invierten en diferentes estrategias

para evitar la depredación, lo que implica diferentes meca-

nismos de defensa. Factores relacionados con el medio

ambiente, la densidad de plantas y/o los rasgos funcionales

de las plantas pueden influir en la herbivoría en los ecosis-

temas acuáticos. Sin embargo, todavía falta información

sobre cómo esos factores influyen en la herbivoría en los

ecosistemas acuáticos y pueden contribuir a la carga de

herbivoría.

Objetivo: Evaluar si existe una relación entre la herbivoría

y las variables ambientales (p. ej., profundidad del agua y

distancia al margen), los factores ecológicos (densidad de

plantas) y los rasgos estructurales de las plantas (altura,

grosor de la hoja y área foliar) e indicar estructuras anató-

micas que actúen junto con los rasgos estructurales en el

sistema de defensa de especies de Montrichardia.

Métodos: Se evaluaron 96 individuos de Montrichardia

spp. (78 de M. arborescens y 18 de M. linifera, en 18

sitios) recolectados en septiembre de 2018. En cada sitio,

se midió la profundidad del agua, la distancia al margen

y la densidad de plantas. De los individuos, medimos la

altura de la planta, el grosor de la hoja y fotografiamos las

hojas para calcular el área foliar específica y la cantidad de

herbivoría (en porcentaje). Para identificar las estructuras

anatómicas relacionadas con la defensa de las plantas, se

recogió hojas completamente expandidas de tres individuos

por cuadrante.

Resultados: Para M. arborescens, las plantas con hojas

más gruesas y mayor área foliar específica tienen menos

daño por herbivoría. Para M. linifera, las plantas con hojas

más gruesas y que habitan en sitios más profundos tienen

más daño por herbívoros, mientras que las plantas más

alejadas del margen tienen menos daño por herbívoros. Se

encontró estructuras anatómicas relacionadas con la defen-

sa, como células idioblásticas con compuestos fenólicos

y células con inclusiones sólidas que pueden contribuir a

evitar daños severos en las características de las hojas.

Conclusiones: Nuestros resultados indican que la herbi-

voría en las especies de Montrichardia podría explicarse

por una combinación de características ambientales (aisla-

miento del parche y profundidad del agua) y de la planta.

Descubrimos que los rasgos de las hojas eran factores

importantes que impulsaban los cambios en la carga de

herbivoría, especialmente el grosor de las hojas y el área

foliar específica. Además, las especies de Montrichardia

invierten en compuestos químicos e inclusiones sólidas

para evitar daños graves en las hojas y, por lo tanto, pueden

sufrir menos daños que otras especies de macrófitos.

Palabras clave: plantas acuáticas; rasgos funcionales;

defensa química; interacciones planta-herbívoro; ecosiste-

mas de agua dulce; Amazonas.

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