Chemosphere 230 (2019) 303e307

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Short Communication

Evaluation of connected clonal growth of Solidago chilensis as an

avoidance mechanism in copper-polluted soils
Felipe Lillo a, Rosanna Ginocchio b, c, **, Christopher Ulriksen a, Elvira A. Dovletyarova d,
Alexander Neaman a, *
a
Escuela de Agronomía, Pontificia Universidad Cato lica de Valparaíso, Quillota, Chile
b lica de Chile, Santiago, Chile
Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Cato
c
Center of Applied Ecology and Sustainability (CAPES), Pontificia Universidad Cato lica de Chile, Santiago, Chile
d
Department of Landscape Design and Sustainable Ecosystems, RUDN University, Moscow, Russia

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Clonal spread was not inhibited in

soils polluted in the uppermost layer
(2 cm depth).

Organic matter eliminated soil cop-
per toxicity allowing normal clonal
spread.

Connected clonal growth is an effec-
tive avoidance mechanism in copper-
polluted soils.

a r t i c l e i n f o a b s t r a c t

Article history: Plant resistance to metals can be achieved by two strategies, tolerance and avoidance. Although metal
Received 26 December 2018 tolerance has been broadly studied in terrestrial plants, avoidance has been less considered as a strategy
Received in revised form to cope with soil metal pollution. Avoidance may be an effective alternative in herbaceous plants with
18 April 2019
connected clonal growth in environments having high heterogeneity in soil micro-spatial distribution of
Accepted 25 April 2019
Available online 1 May 2019
available metals and other soil conditions (i.e. organic matter). In this study, we performed a laboratory
experiment on clonal growth of Solidago chilensis when exposed to copper-spiked soils (800 mg kg1) at
Handling Editor: T. Cutright different depths (0, 2, 5 and 8 cm depth), with (20%) and without addition of organic matter to mimic
contrasting microhabitats found at smelter hinterlands (i.e. open bare ground and microhabitats below
Keywords: shrubs). Results showed that plants grown in the 2 cm-depth Cu-spiked soils were able to growth and
Vegetative growth produce ramets and rhizomes. However, increased Cu uptake of plants determined phytotoxic effects and
Copper smelter a reduction in clonal spread in the 5 cm- and 8 cm-depth Cu-spiked soils. Addition of organic matter to
Soil heterogeneity the Cu-spiked soil layers allowed clonal spread. Considering that ramet and rhizome production is
Metal pollution
decreased but not inhibited when copper pollution is restricted to the uppermost soil layer (2 cm depth)
Copper toxicity
and that organic matter eliminated soil copper toxicity allowing normal clonal spread, connected clonal
growth may be an effective avoidance mechanism of Solidago chilensis, particularly in environments with

* Corresponding author.
** Corresponding author. Departamento de Ecosistemas y Medio Ambiente, Fac-
ultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Catolica de Chile,
Santiago, Chile.
E-mail addresses: rosanna.ginocchio@uc.cl (R. Ginocchio), alexander.neaman@
pucv.cl (A. Neaman).

https://doi.org/10.1016/j.chemosphere.2019.04.199
0045-6535/© 2019 Elsevier Ltd. All rights reserved.
304 F. Lillo et al. / Chemosphere 230 (2019) 303e307

high heterogeneity in micro-spatial distribution of metals and organic matter in the soil profile and
between microhabitats.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction important to state that metal-tolerant plants are scarcely repre-

sented in natural systems, their seeds are hardly available in mar-
Plant resistance to metals can be theoretically achieved by two kets, and they are normally slow growing plants, which produce
strategies, tolerance and avoidance (Do Nascimento and Xing, low biomass (Baker et al., 2010; Mench et al., 2010). On the other
2006; Jutsz and Gnida, 2015; Mickelbart et al., 2015). Although hand, non-metal tolerant herbaceous plants are readily available
metal tolerance has been broadly studied in terrestrial plants, and fast growing. In particular, some plants that can cope with soil
avoidance has been less considered as an effective strategy to cope metal pollution though connected clonal growth. Such plants might
with soil metal pollution. For instance, Solidago chilensis Meyen be a good alternative for phytostabilization of polluted soils
(Asteraceae), a perennial herbaceous plant with connected clonal showing high spatial heterogeneity in soil metal concentrations.
growth with physiological integration, has been found on areas
heavily polluted by a copper smelter in the Puchuncaví valley,
2. Materials and methods
central Chile, reaching up to 12% cover of a total plant cover for the
area of 45e50% (Ginocchio, 1997, 2000). However, this species is
2.1. Experimental design
highly sensitive to increasing copper concentrations under stan-
dard laboratory metal toxicity tests (Ginocchio, 1999).
Solidago chilensis is a native perennial herb species of Chile; this
Soils polluted by atmospheric deposition are known to exhibit
common weed can reach up to 1 m height (Matthei et al., 1995;
high micro-spatial heterogeneity of metal contents and other soil
Russo and Garbarino, 2008; Pereira et al., 2018). Seed collection
characteristics (Nowack et al., 2010; Co rdova et al., 2011). This
procedure and plant propagation are described in detail in the
creates a mosaic of both high and low toxic microhabitats, pro-
Online Supplementary Material. After one month of growth, 10-cm-
ducing safe sites for successful recruitment and growth of non-
height seedlings were individually transferred to experimental
metal tolerant plant species (Ginocchio et al., 2004). Once estab-
boxes, as described below (Supplementary Fig. 1).
lished in safe sites, herbaceous plants with connected clonal
Soil was collected in an area located 5 km to the south-east of
growth may expand along the surface. Physiological integration of
the Ventanas copper smelter, considered as background soil for the
ramets may allow continuous horizontal growth of rhizomes in
area (Ginocchio, 2000). Two parallel laboratory experiments were
high toxic microhabitats with less ramet development, while dense
designed (see Online Supplementary material for details). In the
ramet development may occurs in non-toxic microhabitats, as it
first experiment, we used four treatments comprising 0, 2, 5, and
has been demonstrated at small scales in highly variable nutrient
8 cm depth of Cu-spiked soils laid on top of an 8, 6, 3, and 0 cm
limiting environments (Herben et al., 2015). It has been demon-
depth layer of control soil, i.e., soil without addition of peat nor
strated that clonal growth makes it possible to forage for limited
copper, respectively (Supplementary Fig. 1). The second parallel
resources, share them among connected ramets, and scape sites
experiment followed the same experimental design as in the first
where some limiting resources might have been exhausted
experiment, but the organic matter content of the Cu-spiked soil
(Hutchings and de Kroon, 1994).
layer was increased to 20% with peat (see Online Supplementary
In the particular case of the areas heavily impacted by emissions
material for details). These experimental setups were intended to
of the Ventanas copper smelter in the Puchuncaví valley, strong
mimic soil heterogeneity that exists between described micro-
variability in soil copper concentrations and organic matter content
habitats (below shrub and open bare ground areas between shrubs)
has been found between microhabitats at a distance of meters, such
present at vicinities of the Ventanas copper smelter (Ginocchio
as below shrubs and the open bare ground areas between shrubs
et al., 2004).
(Ginocchio et al., 2004). Specifically, high Cu contents exist on litter
The soil was spiked with 800 mg kg1 of Cu as CuO. This total
and topsoil layers (0e5 cm depth) below shrubs when compared to
soil Cu concentration was equal to that measured in polluted areas
open spaces among shrubs. However, low Cu availability exists due
at vicinities of the Ventanas smelter (Muena et al., 2010); it was
to the high organic matter content found in this microhabitat
above the maximum of 600 mg kg1 measured in areas where
(Ginocchio et al., 2004). Furthermore, it has been demonstrated
Solidago chilensis grows (Ginocchio, 2000) and it was well above the
that metal concentrations are restricted to the uppermost soil layer
phytotoxic levels described for non-metal-tolerant plants (Lindsay
of few centimeters (Ulriksen et al., 2012), while the subsoil remains
and Cox, 1985; Verdejo et al., 2015). This experimental concentra-
unaffected due to low mobility of copper in these soils (Neaman
tion was decided to provide Cu toxicity to plants and therefore to be
et al., 2009).
able to test the hypothesis.
Based on these arguments, we hypothesized that the occurrence
Ten replicate opaque white acrylic boxes (20 cm/10 cm/2 cm;
of Solidago chilensis on heavily copper polluted areas near the
length/height/wide; having three 2-mm diameter holes in their
Ventanas smelter may be explained by avoidance mechanisms
bases) were prepared for each soil treatment, in each experiment
resulting from the capability of connected clonal growth of the
(Supplementary Fig. 2). Even though experimental boxes have
species under highly microhabitat heterogeneity. To test this hy-
small sizes, they were appropriate for the morphology and growth
pothesis, we performed a laboratory experiment on clonal growth
speed of selected plant species, according to the experimental time
of Solidago chilensis, in soils spiked with copper at different depths,
of the present study (Supplementary Fig. 3).
with and without addition of organic matter, mimicking micro-
Before transplanting (one plant per box near an edge of the box),
habitats present in the area (below shrubs and open bare ground
plant roots were cut to a length of 5 cm to homogenize the un-
areas among shrubs).
derground plant material. The experimental plant boxes were
With regards to the significance of the present study, it is
randomly placed into a greenhouse with a 12 h/12 h light/dark
 F. Lillo et al. / Chemosphere 230 (2019) 303e307 305

cycle, mean temperature of 26 ± 5  C, and a maximum light in-
tensity of 3000 Lux. The experimental plant boxes were randomly
reassigned in experimental benches once a week.
2.2. Response variables and data analysis
Dry shoot biomass, shoot height, and dry root biomass of the
original plant, as well as the ramet proliferation (number and dry
biomass) were evaluated after 5 months, in each experiment. Only
in the first experiment, plant tissues were analyzed for Cu as
described in details in the Online Supplementary Material, as they
were the plants that showed copper toxicity symptoms
(Supplementary Fig. 3). Ten replicates were analyzed altogether
due to lack of biomass for the analysis. Thus, it was not possible to
perform statistical analysis of Cu concentrations in the plant
tissues. Likewise, the number of underground rhizomes produced
was monitored only in the first experiment, as ramet production
was not affected after addition of 20% OM (Supplementary Fig. 3).
For each experiment, ANOVA tests were used to compare the
effect of Cu enrichment depth on each response variable. The Tukey
test was used as a posteriori test. All statistical analyses were per-
formed on the Statistica software for Windows (StatSoft Inc., 1993).
3. Results
3.1. Plant growth in soils without addition of organic matter
As mentioned above, plant roots were cut to a length of 5 cm to
homogenize the underground plant material. Thus, plant roots
were completely located in the Cu-spiked layer in the 5- and 8-cm-

Fig. 1. Shoot biomass (A), shoot height (B), root biomass (C), rhizome number (D), ramet biomass (E), and ramet number (F) of Solidago chilensis grown in soils spiked with Cu at
different depths (0, 2, 5, and 8 cm, see text for details). Asterisk means complete inhibition of growth.
306 F. Lillo et al. / Chemosphere 230 (2019) 303e307
depth treatments, while only their upper half was located in Cu-
spiked layer in the 2-cm-depth treatment. For this reason, nega-
tive effects of Cu on plant growth significantly increased with depth
of copper enrichment (Fig. 1A, B, and 1C).
Rhizome proliferation was significantly reduced in the 5-cm-
depth Cu-spiked soils; however, rhizomes were still produced on
the 2-cm-depth Cu-spiked soils (Fig. 1D). Rhizome lengths were
also reduced with increasing depths of Cu enrichment (data not
shown). Complete inhibition in the proliferation of rhizomes
occurred in the 8-cm-depth Cu-spiked soils (Fig. 1D).
Ramet proliferation was still occurring in the 2-cm-depth Cu-
spiked soils (Fig. 1E and F), but plants took longer to start
growing (data not shown). However, complete inhibition of ramet
proliferation occurred in the 5- and 8-cm-depth Cu-spiked soils
(Fig. 1E and F).
Likewise, general plant aspect differed between depths of cop-
per enrichment. In the 5- and 8-cm-depth Cu-spiked soils, plants
showed clear symptoms of toxicity in the upper leaves and had
small and narrow leaves, besides weak tonicity (Supplementary
Fig. 3).
Copper uptake increased with depth of Cu enrichment in Soli-
dago chilensis plants (Supplementary Table 1). Copper concentra-
tions were much higher in roots than in shoots. In the 2 cm- and
5 cm-depth Cu-spiked soils, Cu concentrations in shoots were
below or close the toxic threshold described for most plants
(>50 mg kg1; Adriano (2001); however, in the 5- and 8-cm-depth
Cu-spiked soils, Cu concentrations in roots were above the
threshold described as toxic for most plants (>100e300 mg kg1;
Adriano (2001). An important translocation of Cu from roots to
ramets was detected (Supplementary Table 1) which may have
resulted in toxic effects and thus inhibition of vegetative growth.
3.2. Plant growth in soils with addition of organic matter
Negative effects of Cu on plant biomass were counteracted by
addition of 20% OM, for all depths of Cu enrichment
(Supplementary Table 2). Likewise, ramet number and biomass
were not affected when 20% OM was added to Cu-spiked experi-
mental soils. For this reason, the number of underground rhizomes
was monitored only in the first experiment. Finally, plants did not
show any copper toxicity symptoms in the second experiment
(Supplementary Fig. 3); thus, plant tissues were not analyzed for Cu
in the experiment with addition of organic matter.
4. Discussion
Connected clonal growth has been considered as an effective
avoidance mechanism that allow plants to live in habitats where
great soil spatial heterogeneity exist (Ginocchio, 1994; Alpert et al.,
2003), as physiological integration occurs among interconnected
ramets (Harper, 1977; Bell, 1984; Jo  nsdo
 ttir and Watson, 1997;
Klimes et al., 1997; Ikegami et al., 2008). Ramets explore the soil
surface and adjust its lateral spread according to soil resource levels
(Harper and Bell, 1979; Lovell and Lovell, 1985; Oborny and Kun,
2003). Therefore, ramets established in resource-rich patches
buffer any shortfalls in resource supply among ramets established
in resource-poor patches (Hartnett and Bazzaz, 1985; Marshall and
Price, 1997; Alpert et al., 2003), resulting in a net benefit to the
whole plant (Cook, 1983).
However, the above-mentioned plant growth strategy has not
been described in metal-polluted habitats with great soil spatial
heterogeneity. In this study, we have experimentally demonstrated
its efficacy in S. chilensis. Specifically, S. chilensis is clonal plant
species with connected growth, such as S. altissima and S. gigantean
nsdo
(Jo ttir and Watson, 1997). It is a non-metal tolerant species and
therefore no metal tolerance mechanisms are involved. However,
our results support that ramets established in non-metal polluted
soil patches could support growth of ramets on high-metal polluted
soil patches through physiological integration, thus avoiding metal
toxicity.
With regards to organic matter addition in Cu-spiked soils, we
assume that organic matter reduced Cu phytoxicity as shown in
literature. Specifically, added organic matter is known to produce
organic acids that form complexes with Cu in the solution, resulting
in the decrease of the Cu2þ ion activity (Goecke et al., 2011), which
is considered the most phytotoxic form of Cu in the soil (Sauve 
et al., 1998; Thakali et al., 2006). Likewise, organic matter is
known to bind Cu ions on sorption sites on the solid phase
(Mondaca et al., 2015). However, we cannot provide direct evi-
dences of decrease of metal uptake, since analysis of plant tissues
was performed only in the case of the first experiment without
organic matter addition.
5. Conclusion
Results of the present study confirm the hypothesis that the
occurrence of Solidago chilensis, a non-metal tolerant plant, on an
area heavily polluted by copper may be explained by avoidance
mechanisms resulting from the capability of connected clonal
growth of the species under high micro-spatial heterogeneity of
copper content and other soil characteristics (i.e. organic matter).
Possible explanations for this phenomenon are ramet proliferation
in organic matter-rich patches, such as microhabitats below shrubs,
and/or rhizome proliferation below the topsoil layer of few centi-
meters depth, which is the most rich in metals.
Acknowledgements
The authors thank the field assistance of Francisco Cabrera and
Gaston Carvallo, and the laboratory assistance of Margarita Cam-
sar Verdugo, Daniela Castro, Carolina Lazcano and Ignacia
pos, Ce
Toro. This study was funded by project FONDECYT 1000750 to
Rosanna Ginocchio. Article writing was supported by the CONICYT
PIA/BASAL FB0002 project (Center of Applied Ecology and Sus-
tainability, CAPES) and the “5e100” project of RUDN University,
Russia.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.chemosphere.2019.04.199.
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