ORIGINAL RESEARCH

published: 18 December 2018

doi: 10.3389/fpls.2018.01835

Allelopathic Effect of the Invasive

Ludwigia hexapetala on Growth of
Three Macrophyte Species
Gabrielle Thiébaut 1* , Lise Thouvenot 2,3 and Hector Rodríguez-Pérez 1
1
ECOBIO, UMR 6553 CNRS, Université de Rennes 1, Rennes, France, 2 German Centre for Integrative Biodiversity
Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany, 3 Institute of Biology, Leipzig University, Leipzig, Germany

The release of allelochemicals by plants can affect the performance of other organisms
positively or negatively. We tested the effects of aqueous extracts and leachates derived
from the leaves and roots of the invasive water primrose (Ludwigia hexapetala) on one
submerged native species – Ceratophyllum demersum, and two exotic species – the
submerged Egeria densa and the emergent growth form of Myriophyllum aquaticum.
The effect of the aqueous extracts and leachates of L. hexapetala on photosynthetic
yield, growth (i.e., relative growth rate, leaf area), root length, and length of the
lateral shoots of each species were analyzed in spring and in autumn. In autumn,
an allelopathic effect was established on the traits of the three macrophytes species.
The root extracts stimulated leaf area and the photosynthetic yield of C. demersum
Edited by:
Te Cao, and of E. densa, whereas leaf treatments (leachates and extracts) and root leachate
Chinese Academy of Sciences, China reduced the leaf area of M. aquaticum. The autumnal root leachate of L. hexapetala
Reviewed by: decreased the relative growth rate of C. demersum, whereas it had no effect on
Yu Cao,
Wuhan Botanical Garden (CAS),
the two others plants. The root extract increased the length of lateral branches of
China M. aquaticum in autumn, suggesting a positive effect of L. hexapetala on the lateral
Aiping Wu,
growth of M. aquaticum. Three main allelochemicals were identified in leaves: quercitrin,
Hunan Agricultural University, China
prunin, myricitrin. The concentrations of these allelochemicals were greater in the leaf
*Correspondence:
Gabrielle Thiébaut extract taken from L. hexapetala in autumn than in spring, and those found in the
[email protected] leaf leachates for both seasons. This assessment of autumnal allelopathy could help
to explain the patterns of plant community succession in invaded areas.
Specialty section:
This article was submitted to Keywords: allelopathy, macrophytes, native, exotic species, functional traits
Functional Plant Ecology,
a section of the journal
Frontiers in Plant Science
INTRODUCTION
Received: 18 July 2018
Accepted: 27 November 2018 The allelochemicals released by organisms into the environment, also called “allelopathy” (Rice,
Published: 18 December 2018
1984; Elakovich and Wooten, 1989) have beneficial or detrimental effects on neighboring organisms
Citation: (e.g., phytotoxicity, soil sickness). Allelochemicals are released directly from plants through
Thiébaut G, Thouvenot L and different mechanisms, such as root exudation, leaching of aerial parts, and volatilization, and also
Rodríguez-Pérez H (2018) Allelopathic
passively through plant decomposition. The role of allelopathy on the structure and composition of
Effect of the Invasive Ludwigia
hexapetala on Growth of Three
biological communities is relatively unexplored in freshwaters (Kulshretha and Gopal, 1983; Agami
Macrophyte Species. and Waisel, 1985; Elakovich and Wooten, 1989; Gross, 2003; Dandelot et al., 2008). Nevertheless,
Front. Plant Sci. 9:1835. some studies showed that several aquatic macrophytes (i.e., Elodea nuttallii, Myriophyllum
doi: 10.3389/fpls.2018.01835 spicatum) can impact the phytoplankton (Gross, 2003) and inhibit germination and/or seedling

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

growth (Gopal and Goel, 1993; Dandelot et al., 2008) via the three target species and (2) that the effects of L. hexapetala on
release of allelochemicals. Bioassays, using plant extracts (i.e., the target plants would change according to the season.
leachates, exudates), are one of the most common methods used
to assess the allelopathic effects of plants. Overall, they have
generally only tested the allelopathic potential of plants at one MATERIALS AND METHODS
point in time, even though the synthesis of allelochemicals and
their concentrations in the plant fluctuate throughout the year Plant Materials
(Helmig et al., 2013; Santonja et al., 2018). Indeed, the seasonal The amphibious Parrot’s Feather, M. aquaticum, native to South
variation in allelochemicals could be explained by the fluctuations America was introduced as ornamental plant into France in
of abiotic and biotic parameters, i.e., climatic conditions (Petrussa approximately 1880 (Sheppard et al., 2006). The species can cause
et al., 2013), the presence of herbivores and/or pathogens (Gatti severe problems in Southern Europe (Les and Merhoff, 1999),
et al., 2014; Silva et al., 2014) and stage in the life-history of the in the southern states of the United States, in South America
plant (Lombardo et al., 2013; Santonja et al., 2018). The seasonal (Fernandez et al., 1993), in New-Zealand and Australia. Once
dependence of plant allelopathic interactions is still understudied, introduced into a new region it spreads rapidly, primarily by
although it could help to explain exotic plant establishment, vegetative stem fragmentation. It is often found in eutrophic
their spread and plant community succession in invaded areas. water bodies (small streams, ponds, slow-running waters and
Indeed, the potential allelopathy of exotic plants could favor irrigation channels). Stems of M. aquaticum float out over the
their establishment and their spread into their introduced water surface to form dense mats from which emergent shoots
range (Callaway and Ridenour, 2004). Many exotic plants could arise (Hussner, 2009). This species has demonstrated a potential
synthesize unknown allelochemicals and release them into the inhibitory effect on neighboring plants (Elakovich and Wooten,
native community (c.f. “Novel Weapons Hypothesis,” Callaway 1989).
and Ridenour, 2004). These novel allelochemicals could inhibit The Brazilian water-weed, E. densa, is a native, submerged
the growth of native plants and thus improve the growth of the macrophyte coming from Argentina, Brazil, and Uruguay.
invasive species (Callaway and Ridenour, 2004; Kim and Lee, Historically, this species was introduced outside its native range
2011). In this way, as stipulated in the Invasional Meltdown by the aquarium trade. It has been in cultivation in France at
Hypothesis (Simberloff and Von Holle, 1999), the introduction least since 1919. It was observed in the field in France in 1960
of one species may favor the introduction and spread of one or (Cook and Urmi-König, 1984) and is considered as a nuisance
more other exotic species. However, they can also affect other in Central and North America, in Europe and in Australasia
invasive species negatively, if they do not come from the same (Cook and Urmi-König, 1984). E. densa reproduces vegetatively
biogeographical area. from plant fragments. It has a massive build-up of biomass,
This paper is focused on the potential allelopathic effect of the allowing it to become highly invasive. Its dense mats reduce
invasive water primrose Ludwigia hexapetala (Hook. and Arn.) recreational activities and crowd out native species, as well
Zardini, H. Y. Gu and P. H. Raven. (syn. L. grandiflora subsp. as altering the hydrology. Several authors (Nakai et al., 1999;
hexapetala) on three macrophytes species. The amphibious Vanderstukken et al., 2011; Espinosa-Rodríguez et al., 2016) have
L. hexapetala, native to South America, was introduced to found allelochemicals which affect phytoplankton negatively.
Southern France in 1830 (Thouvenot et al., 2013a). Once The European Coontail (C. demersum) is a rootless submerged
established, it formed dense mats in freshwaters, on riverbanks plant found in freshwaters with moderate to high nutrient levels.
and in meadows (Thouvenot et al., 2013a). Its invasive success This plant drifts in the water without being attached to the
could be partially explained by the release of secondary sediment and the species is usually well equipped to capture high
metabolites into the recipient community (Dandelot et al., 2008; to very high nutrient levels from the surrounding water (Denny,
Santonja et al., 2018) which could limit the growth of native 1972). According to several authors (Kleiven and Szczepańska,
plants. Indeed, the presence of L. hexapetala reduces both the 1988; Elakovich and Wooten, 1989). C. demersum contains
plant richness and the abundance of the native species such allelopathic compounds. Aqueous extracts showed inhibitory
as the submerged Ceratophyllum demersum or some emergent effects on seed development of Lepidium sativum (Kleiven and
species (Alisma plantago-aquatica, Lycopus europaeus; Stiers Szczepańska, 1988) and on seedling radicle growth of lettuce
et al., 2011). The water primrose L. hexapetala exhibits a (Elakovich and Wooten, 1989).
horizontal growth stage over water with small round leaves All the target species are macrophytes with an allelopathic
and a growth stage with erect elongated leaves. We used root potential. To avoid a history of interactions between the target
and leaf leachates and aqueous extracts of L. hexapetala from species and the water primrose, the target species E. densa,
individuals collected in spring and in autumn to analyze the M. aquaticum, and C. demersum were bought in a garden center,
impact of these solutions on the traits of one native species whereas L. hexapetala was collected in the field. In this way, the
[C. demersum L. (Ceratophyllaceae)], and on two exotic species target plants were considered “naïve” to the water primrose.
[Myriophyllum aquaticum (Vell.) Verdc. (Haloragaceae)], and
[Egeria densa Planch. (Hydrocharitaceae)]. Our aim was to gain Methods
a better understanding of the responses of other macrophytes Preparation of the Treatments
species to the invasive species. We hypothesized (1) that leaf and For this study, 100 g of small round leaves and 100 g of
root treatments would induce a decrease in the growth of the sediment roots of L. hexapetala were collected from a pond at

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

Apigné in Brittany, France (01◦ 440 25.200 O, 48◦ 050 41.400 N), in monitoring photosynthetic activities. The fluorescence yield was
spring at mid-March and in autumn at the end of September. measured on the apex leaves using a Diving-PAM underwater
The leaves and roots of L. hexapetala were washed to remove fluorometer (Walz) after a 30-min period of dark adaptation in
zooplankton and epiphytes. Aqueous extracts of live leaves and the afternoon of the first day and then every week for 28 days.
sediment roots were prepared in tap water by crushing 100 g The initial fluorescence – Fo – and maximal fluorescence – Fm –
of fresh leaves or fresh roots in 2000 mL of tap water, and the were recorded by turning on the weak measuring light, and Fm
pulpy mixture was stored for 72 h at 4◦ C. The mixture was was given after the saturation flash. The maximum quantum yield
filtered through filter paper (Whatman #1) to remove smaller (Fv/Fm) was calculated as Fv/Fm = (Fm−Fo)/Fm with Fv variable
particulate matter (Elakovich and Wooten, 1989). Then it was Fluorescence.
centrifuged for 15 min at 9,000 rpm. The supernatant thus Four morphological traits (plant stem length, roots and lateral
obtained constituted the aqueous extract. The leaf and root branches length, and leaf area) of each plant were measured after
leachates of L. hexapetala were prepared by soaking 100 g (fresh 28 days of exposure to the aqueous extracts or leachates solutions
leaves or fresh roots) in 2000 mL of tap water for 72 h in the dark in the laboratory. One picture was taken of one leaf per plant at
at 4◦ C. They were then filtered through filter paper (Whatman the beginning and at the end of the experiment. The leaf area was
#1). Each leachate/aqueous extract was divided into two: one part measured using Image J software. The relative growth rate (RGR;
(1500 mL) was used to test the potential allelopathic effect of cm d−1 ) was calculated following Hunt’s (1990) formulation:
L. hexapetala on the three macrophytes species and the second RGR = [ln(L2)-ln(L1)]/(T2-T1), in which L1 and L2 refer to stem
(500 mL) was used to identify allelochemicals. length at time points T1 and T2. The same experimental design
was applied in spring and in autumn.
Experimental Design
The individuals of E. densa, M. aquaticum, and C. demersum Chemical Composition of the Leaf/Root
were bought in a garden center 15 days before the beginning
Aqueous Extracts and Leachates of
of the experiment in spring and in autumn (respectively,
at the beginning of March and in mid-September) and L. hexapetala
acclimatized in tap water at the ambient temperature for 2 weeks. The leaf leachates and leaf aqueous extracts of L. hexapetala that
The tap water was slightly basic with a moderate nutrient were not used in the allelopathy experiment, were lyophilized
concentration (mean annual value according to French Water and ground into a powder prior to chemical analysis. The leaf
Agency data: conductivity = 462 µS cm−1 ; pH = 7.95; [NO3 − leachates and aqueous extracts of L. hexapetala in spring and in
N] = 6.95 mg L−1 ; [NH4 + N] = 0.03 mg L−1 ; [PO4 3− autumn were analyzed using liquid chromatography and high
P] = 0.043 mg L−1 ). The amphibious M. aquaticum had both resolution mass spectrometry (LCMS) according to the method
submerged leaves and aerial leaves which emerged above the described by Santonja et al. (2018). There was not enough root
surface of the water. After the acclimatization period, the three material after lyophilisation to conduct the analyses.
target plants – C. demersum, M. aquaticum, and E. densa – were
rinsed with distilled water and their shoots cut to a length of Statistical Analysis
5 cm. All the selected shoots had an intact apex, no roots, and Photosynthetic yields were analyzed on a repeated measures basis
no trace of necrosis, buds, or lateral stems. One shoot of each using a non-parametric test (Naguchi et al., 2012), since the data
plant species (E. densa, C. demersum, and M. aquaticum) was did not meet homoscedasticity and normality requirements for
placed in a cylindrical plastic tube (100 mL, height: 10 cm) filled parametric tests. Whenever treatment effect or the interaction
with 50 mL of the solution (i.e., leaf or root leachates, or leaf between treatment and time was significant, a pairwise
or root extracts, or tap water). The water level in each plastic comparison of treatment levels and treatment levels within a
tube was maintained by adding tap water, to avoid increasing given sampling time was performed using a Mann–Whitney–
allelochemical concentrations, plant desiccation and nutrient Wilcox test, and a Benjamini-Hochberg False Discovery Rate
deficiencies and to offset losses from evaporation. Ten replicates (10% acceptance level) correction was subsequently applied
were used per plant species and treatment. Plants were placed in to multiple test series (Benjamini and Yekutieli, 2001). Non-
one growth chamber (Photon Flux Density 80 µmol s−1 m2 , 12 h parametric repeated measures analyses were performed with R
light/12 h dark cycle, and at 16◦ C) for 28 days, and their position software (R Core Team, 2016) and a nparLD package (Naguchi
in the chamber was completely randomized. The incubation et al., 2012).
temperature of 16◦ C was the maximal temperature observed in The abilities of each plant species to grow and produce roots
spring and autumn. and lateral branches under different treatments depending on
the season were analyzed using a two-way linear model. The leaf
Measured Variables area growth of C. demersum, as well as the length of the lateral
Photosynthetic yield was monitored to assess the impact shoots and roots of each species were log-transformed to check
of allelochemical stress responses on the photosynthesis of their residual homoscedasticity and normality. When the number
the target plants. To evaluate the allelopathic effect on the of data available per trait and combination of season treatments
photosynthetic yield, a pulse amplitude modulation (PAM) was strictly lower than three, the combination was not included
chlorophyll fluorometer was used to measure photosynthetic in the statistical analysis dataset. This was particularly the case
activity. PAM is a convenient and sensitive method for for the lateral shoots and root length, as the species did not

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

produce lateral shoots or roots in every treatment. Consequently, p < 0.0001, Figure 2A), although it was not impacted by the
some treatments do not appear in the results section or in the treatments (Table 2). Leaf area growth was affected negatively
Figures and Tables in this paper. The adequate distribution of in autumn by the leaf treatments and by the root leachates
model residuals was verified for each trait by checking the model (F = 3.17; p = 0.018, Figure 2B). The lengths of lateral shoots
plots. Tukey’s HSD tests were applied to observe differences of M. aquaticum were longer after exposure to the root extracts
between treatments. Untransformed means and standard errors (F = 3.89; p = 0.027, Figure 2C) than after exposure to leaf
were used in the Figures to facilitate interpretation. Statistical treatments.
analyses were performed with R software (R Core Team, The autumnal root treatments and the leaf leachates
2016). stimulated the photosynthetic yield of E. densa after 7 and
28 days in the plants exposed to the root extracts (interaction
treatment × sampling date, Table 1 and Figures 3C,D). RGR
RESULTS and leaf area growth of E. densa were affected by the interactions
between the treatment and the season (Table 2), but the length
There were no significant effects of spring aqueous extracts and of the roots only depended on the season (Table 2). The
leachates on the photosynthetic yield or on the morphological autumnal root extract stimulated the growth of the E. densa leaves
traits of M. aquaticum, E. densa, and C. demersum (Tables 1, 2 (interaction season × treatment: F = 6.02; p = 0.0003, Figure 3B).
and Figures 1–4). Moreover, aqueous extracts and leachates The lengths of the roots (F = 150.27; p < 0.0001, Figure 3D) were
had no significant effect on the photosynthetic yield of higher in autumn than in spring (Table 2).
M. aquaticum in autumn (Table 1 and Figures 1A,B). The RGR The photosynthetic yield of C. demersum was stimulated by
of M. aquaticum was higher in autumn than in spring (F = 195.51; the root extracts in autumn (Table 1 and Figure 1F). Relative

TABLE 1 | Effects of leachates and extracts of Ludwigia hexapetala on the photosynthetic yield of target species in spring and in autumn.

Autumn Spring

ATS df p ATS df p

Myriophyllum aquaticum Treatment 0.73 3.23 0.6 0.96 2.37 0.4

Time 39.67 3.02 <0.0001 39.18 3.30 <0.0001
Treatment × time 1.14 8.75 0.3 1.65 8.03 0.1
Egeria densa Treatment 16.11 3.85 <0.0001 0.19 3.88 0.9
Time 40.56 3.58 <0.0001 25.21 2.86 <0.0001
Treatment × time 2.68 10.66 0.002 1.24 7.81 0.3
Ceratophyllum demersum Treatment 5.09 2.66 0.003 1.94 3.31 0.1
Time 6.67 1.69 0.002 11.93 2.43 <0.0001
Treatment × time 1.20 2.01 0.3 1.4 3.5 0.2

The significant differences are indicated in bold type; ATS denotes ANOVA Type Statistic.

TABLE 2 | Summary of the two-factor linear model, with season and treatment as factors, on the different morphological traits measured for each species
(C. demersum, E. densa, and M. aquaticum) in the laboratory experiment (with the degrees of freedom (Df), the range of the number of replicates per modalities for each
factor (n), F-values for each factor per trait, and the significance level (p).

Relative growth rate Leaf area growth Length of lateral shoots Length of roots

Df n F p n F p n F p n F p

C. demersum
Season 1 31–50 0.55 0.46 29–50 2.00 0.16 / / / / / /
Treatment 4 10–20 2.30 0.07 10-20 1.19 0.32 / / / / / /
Season × treatment 3 6–10 3.54 0.02 4–10 2.72 0.051 / / / / / /
E. densa
Season 1 46–50 55.92 <0.0001 44–50 12.31 <0.001 11–34 3.03 0.09 31–35 150.27 <0.0001
Treatment 4 16–20 4.57 0.002 16–20 3.26 0.015 6–12 1.35 0.27 6–19 2.51 0.052
Season × treatment 4 6–10 4.84 0.001 6–10 6.02 <0.001 3–8 0.48 0.62 3–10 1.98 0.13
M. aquaticum
Season 1 42–48 195.51 <0.0001 40–49 35.08 <0.0001 / / / 23–27 1.43 0.24
Treatment∗ 4 15–20 1.39 0.25 16–20 3.30 0.015 4–7 3.70 0.03 7–14 0.87 0.49
Season × treatment 4 6–10 0.26 0.90 6–10 3.17 0.02 / / / 3–9 0.62 0.65

Significant results are in bold type; tendencies are in italic. ∗ Df = 3 for the length of lateral shoots.

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

FIGURE 1 | Mean photosynthetic yield plus standard error for the three species in spring and autumn: Myriophyllum aquaticum (A in spring and B in autumn), Egeria
densa (C in spring and D in autumn), and Ceratophyllum demersum (E in spring and F in autumn). Treatments are denoted by white squares for leaf extract
treatments, black squares for root extract treatments, white triangles for leaf leachate treatments, black triangles for root leachate treatments, and black circles for
control series. Symbols marked with the same letter are not significantly different for treatment factors within the same period (p > 0.05), according to
Mann–Whitney–Wilcoxon pairwise comparisons significance test adjusted by the Benjamini-Yekutieli correction.

growth rate (RGR) of C. demersum was particularly affected by extracts than in the leaf leachates (Table 3). They were higher in
the interaction season × treatment (F = 3.54; p = 0.019, Table 2). autumn than in spring (Table 3).
Indeed, the root leachates of L. hexapetala decreased the RGR of
C. demersum in autumn (Figure 4A). In contrast, the growth of
the leaf area tended to be higher when C. demersum was exposed
to the root treatments than the control treatment in autumn
DISCUSSION
(interaction season × treatment: F = 2.72; p = 0.051, Figure 4B).
The Coontail (C. demersum) produced no roots and very few
Potential Allelopathic Effects of
lateral branches both in spring and autumn (Table 2). L. hexapetala on the Growth of Other
Three main allelochemicals were identified in the leaf leachates Macrophyte Species
and extracts of L. hexapetala: quercitrin, prunin, myricitrin. This study aimed to assess whether the aqueous leaf/root extracts
Concentrations of these allelochemicals were greater in the leaf or the leaf/root leachates of the L. hexapetala had a positive

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

FIGURE 2 | (A) Relative growth rate, (B) leaf area growth, (C) length of lateral shoots, and (D) length of roots of M. aquaticum (mean ± SE) in spring (white symbols)
and autumn (black symbols) depending on the treatment (i.e., control, leaf leachates, leaf aqueous extracts, root leachates, root aqueous extracts) after a 28-day
experiment in the laboratory. Different small letters indicate significant differences between the interaction season × treatment. Stars show significant differences
between seasons.

FIGURE 3 | (A) Relative growth rate, (B) leaf area growth, (C) length of lateral shoots, and (D) length of roots of E. densa (mean ± SE) in spring (white symbols) and
autumn (black symbols) depending on the treatment (i.e., control, leaf leachates, leaf aqueous extracts, root leachates, roots aqueous extracts) after a 28-day
experiment in the laboratory. Different small letters indicate significant differences between the interaction season × treatment. Stars show significant differences
between seasons.

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

FIGURE 4 | (A) Relative growth rate and (B) leaf area growth of C. demersum (mean ± SE) in spring (white symbols) and autumn (black symbols) depending on the
treatment (i.e. control, leaf leachates, leaf aqueous extracts, root leachates, root aqueous extracts) after a 28-day experiment in the laboratory. Different small letters
indicate significant differences between the interaction season × treatment.

or negative effect on the development of other macrophyte indirectly affect its development by modifying the chemical and
species. Our results showed that the root extracts stimulated physical properties of the soil/sediment and by regulating the
the lateral branches of M. aquaticum and the leaf area of the soil microbial community (Walker et al., 2003). Parrot’s Feather
two submerged species C. demersum and E. densa. Conversely, (M. aquaticum) may have the capacity to protect itself from
the leaf treatments and root leachates inhibited the leaf area allelopathy by metabolizing certain phenolic compounds with
growth of M. aquaticum. The latter result could be explained allelopathic activity (Elakovich and Wooten, 1989). This ability
by similar biological type and niche overlap: L. hexapetala and of Parrot’s Feather to synthesize phenols gives it an advantage
M. aquaticum are both exotic species with similar growth forms in allelopathic interactions and may favor its competitiveness.
(amphibious) native from the same geographical range. Although Further studies testing the effect of M. aquaticum root/leaf
the two species co-exist in the field, their patches are spatially leachates on L. hexapetala are required.
separated. In a previous study, we found that L. hexapetala However, we observed that the effects of the root and leaf
stimulated the root production and the growth of M. aquaticum treatments differ according to the biological growth forms of
at low densities (Thouvenot et al., 2013b). The allelochemicals the exotic species. The biological type of plants affects levels
released by roots of L. hexapetala could directly favor the lateral of secondary compounds. For example, emergent species in
growth of M. aquaticum (length of lateral shoots) or could also wetlands contain more phenolics than submerged plant species

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

TABLE 3 | Mean concentrations (± SE; n = 3; µg L−1 ) of chemical compounds produced by short leaves is dependent on the season (Dandelot
found in the leaf aqueous extracts and leachates of Ludwigia hexapetala in spring et al., 2008). The seasonal fluctuations could alter the allelopathic
and in autumn.
activity of the secondary compounds (Santonja et al., 2018). Our
Spring Autumn results should be used with caution because the different impact
of aqueous extracts and leachates according to the season could
Leaf leachates
be explained both by seasonal fluctuations in the physiology of
Myricitrin 88.0 +/− 9.1a 334.4 +/− 9.2b
the target species (C. demersum, E. densa, M. aquaticum) and
Prunin 56.6 +/− 0.6a 121.5 +/− 3.4b
of the donor species L. hexapetala. However, our target species
Quercitrin 97.7 +/− 1.7a 141.9 +/− 4.1b
are clonal individuals, cultivated under glasshouse, conditions
Leaf aqueous extract
which should reduce the fluctuations due to plant phenology.
Myricitrin 2538a 8899b
Furthermore, the absence of a seasonal fluctuation in the RGR
Prunin 2185a 3187b
of E. densa and of C. demersum in the control suggested that
Quercitrin 2877a 3981b
the seasonal fluctuation of donor species L. hexapetala is the
One-way ANOVAs were performed for differences between seasons for each basis of potential seasonal effects of the allelopathy. The growth
chemical compound. F-values and associated P-values are indicated. Different
letters denote significant differences between the two seasons with a < b (post hoc
rate of M. aquaticum was not affected by allelopathy but only
Tukey tests results after one-way ANOVA); d.f., degree of freedom. by season. The ability of M. aquaticum to metabolize phenolic
compounds could counteract the impact of high concentrations
of allelochemicals produced by the water primrose in autumn.
(Smolders et al., 2000). In our experiment, there was no effect of
treatments on the apical growth (RGR) of E. densa, a species from
the same geographical area as the water primrose. Previously, we Chemical Composition of the Leaf/Root
had established that there was no competition between E. densa Aqueous Extracts and Leachates of
and L. hexapetala, but that there was facilitation (Thouvenot L. hexapetala
et al., 2013b). The secondary compounds produced by the roots of Three main flavonoids belonging to the polyphenol family were
the water primrose stimulated leaf area growth and consequently identified in the leaf treatments in spring and in autumn:
had a positive impact on the photosynthetic yield. Our results quercitrin, prunin, myricitrin. The allelochemical composition is
suggested positive interactions between the water primrose and phylogenetically determined (Grutters et al., 2017). Numerous
the submerged E. densa. In contrast, the growth of the native compounds are produced by Ludwigia sp.: saponins, tannins,
submerged plant C. demersum was inhibited by root leachates. polyphenols, alkaloids, linoleic acids, and flavonoids (Averett
This result is congruent with the “Novel Weapons Hypothesis” et al., 1990). However, phenolics are the compounds most
(Callaway and Ridenour, 2004) and with the literature (Sakpere frequently involved in allelopathy in freshwaters (Gross, 2003;
et al., 2010). Previously, Kulshretha and Gopal (1983) established Iason et al., 2013) or between aquatic plant species (Dandelot
that C. demersum and C. muricatum were negatively affected et al., 2008). According to an analysis of root extract conducted
by the exotic submerged Hydrilla verticillata. Moreover, Sakpere by Marcellin-Gros (2015), the most abundant secondary
et al. (2010) observed that exudates of Ludwigia decurrens and compounds in L. hexapetala are two tannins (pedunculagin and
Ludwigia adscendens reduced the stem length of Corchorus an ellagic acid) and the flavonoid quercetin. The result found
olitorius seedlings in early growth. by Marcellin-Gros (2015) suggested that composition of the root
extracts differed from that of the leaf treatments, except for
Seasonal Effect of L. hexapetala on the quercitrin. Quercitrin had a positive effect on the photosynthetic
Other Macrophyte Species yield of phytoplankton (Santonja et al., 2018), and was higher in
The autumnal treatments had significant effects on the the root extracts of L. hexapetala (Marcellin-Gros, 2015) than in
photosynthetic yield and on the morphological traits of the two leaf extracts. This secondary compound may have been released
submerged species and the exotic amphiphyte, whereas no spring into the water by the roots of the water primrose and could have
effects were established. The variation in significance of the effects stimulated the photosynthetic yield and the leaf area of the two
of L. hexapetala, depending on the time at which the roots submerged species E. densa and C. demersum. Previous work has
and the leaves were collected, confirmed the hypothesis that the suggested that the tannin pedunculagin is characterized by its
plants change seasonally. Previous studies have also reported a antioxidant properties and its positive effects on human health
seasonal pattern of allelopathic interactions (Bauer et al., 2009; (Biswas et al., 2014), whereas ellagic acid is a rooting inhibitor
Silva et al., 2014). For example, Bauer et al. (2009) showed (Viéitez and Ballester, 1986; Qin et al., 2006). However, there
an optimal inhibitory effect of Myriophyllum verticillatum on was no root inhibition detected for E. densa and M. aquaticum.
the cyanobacteria Anabaena variabilis in spring. Indeed, the Thus, the roles of these two tannins are unknown and need to be
secondary compound composition in L. hexapetala could be investigated further.
different in spring and autumn (Dandelot et al., 2008; Santonja The concentrations of the allelochemicals in the leaves were
et al., 2018) due to variations in the environmental conditions higher in autumn, which may be related to the seasonal
of the Apigné ponds (i.e., nutrients, light intensity or water fluctuations of environmental parameters and trade-offs between
depth), of climatic parameters (Chaves and Escudero, 1999) and primary and secondary metabolisms. In autumn, plants are
of the plant phenological stage. The presence of allelochemicals exposed to a decrease in temperatures and solar radiation, which

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Thiébaut et al. Allelopathic Effects of Water Primrose on Macrophytes

has been reported to have an inverse relationship with secondary defense (Herms and Mattson, 1992). The variation in the effects
metabolite accumulation in plant tissues (Silva et al., 2014). of L. hexapetala demonstrated in this study highlights the
importance of conducting allelopathy research during different
seasons; if this variation is not considered, the results may not
CONCLUSION reflect the potential effect of a plant species correctly (Silva et al.,
2014). Moreover, the seasonal dependence of biotic interactions
The first hypothesis of this study was partially validated for
has not been studied in depth and it is necessary to take this
M. aquaticum and for C. demersum, indicating an autumnal
into account in order to gain a better understanding of the
phytotoxic effect of leaf treatments on the leaf area growth of the
interactions between native and exotic macrophyte species and
exotic Parrot’s Feather and of root leachates on the growth of the
between different exotic species.
native species. However, L. hexapetala favors the growth of lateral
shoots of M. aquaticum and the leaf area and photosynthetic yield
of E. densa; suggesting a facilitation effect of the root treatment
on the two other exotic species. In contrast, L. hexapetala AUTHOR CONTRIBUTIONS
strongly reduced the growth of the native plant. However, many
plants release allelochemicals into the environment with little GT and LT designed and conducted the experiments. LT
impact on the performance of native plants, due to a long and HR-P analyzed the data. GT wrote the manuscript with
coevolutionary history (Thorpe et al., 2009). Indeed, there are contributions from LT and HR-P.
several biotic and abiotic factors (interactions between plants
and herbivores/pathogens, climatic conditions) that are able to
change allelopathic impact on the recipient community (Inderjit ACKNOWLEDGMENTS
et al., 2011).
Our second hypothesis, that the impact of leaf and root The researchers would especially like to thank Camille Puech
treatments on the growth of the three target species showed and Laurent Martinez for their assistance during laboratory
seasonal fluctuations, was validated. There was no impact of experiments, Mathieu Santonja for chemical analysis, and
the treatment in spring. Plant growth is optimal in spring. Nathalie Marnet for assistance during the chemical analyses.
There are trade-offs between the primary biological functions Chemical analyses were performed at the Plateau de Profilage
of plants, such as growth, and resource allocation for chemical Métabolique et Métabolomique (P2M2, INRA, Le Rheu, France).

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Sakpere, A. M., Oziegbe, M., and Bilesanmi, I. A. (2010). Allelopathiceffectsof Copyright © 2018 Thiébaut, Thouvenot and Rodríguez-Pérez. This is an open-access
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functional implications of macrophyte-phytoplankton allelopathic interactions. No use, distribution or reproduction is permitted which does not comply with these
Freshw. Biol. 63, 1161–1172. doi: 10.1111/fwb.13124 terms.

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