Effects of Shade on Individual Ramet Growth and on Clonal

Growth of the Aquatic Fern Salvinia auriculata (Salviniaceae)

Author(s): Jessica Cristina Carvalho Medeiros, Josiane Carvalho Fonseca Silva,
Grazielle Sales Teodoro, and Flávia de Freitas Coelho
Source: American Fern Journal, 107(1):21-29.
Published By: The American Fern Society
DOI: http://dx.doi.org/10.1640/0002-8444-107.1.21
URL: http://www.bioone.org/doi/full/10.1640/0002-8444-107.1.21

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American Fern Journal 107(1):21–29 (2017)
Published on 24 May 2017

Effects of Shade on Individual Ramet Growth and on

Clonal Growth of the Aquatic Fern Salvinia auriculata
(Salviniaceae)
JESSICA CRISTINA CARVALHO MEDEIROS
Laboratory of Evolutionary Ecology of Herbaceous Plants, Department of Biology, Universidade
Federal de Lavras, zip code 37200-000, Lavras, MG, Brazil, e-mail: [email protected]

JOSIANE CARVALHO FONSECA SILVA

Laboratory of Evolutionary Ecology of Herbaceous Plants, Department of Biology, Universidade
Federal de Lavras, zip code 37200-000, Lavras, MG, Brazil, email: [email protected]

GRAZIELLE SALES TEODORO

Institute of Biology, Universidade Estadual de Campinas, Monteiro Lobato street, 255, Cidade
Universitária, zip code 13083-862, Campinas, SP, Brazil, e-mail: [email protected]

FLÁVIA DE FREITAS COELHO

Laboratory of Evolutionary Ecology of Herbaceous Plants, Department of Biology, Universidade
Federal de Lavras, zip code 37200-000, Lavras, MG, Brazil, e-mail: [email protected]

ABSTRACT.—Light intensity is among the major environmental factors determining the ecological
distribution of any plant. The effects of light availability on aquatic plants continue to be
discussed, as some species show higher growth under full sun conditions and other species show
reduced growth. The aim of this study was to test the effects of different shade levels on ramets and
clonal growth of the fern Salvinia auriculata. We hypothesized that the ramets grow better under
shade conditions over time as in most land ferns. We performed a greenhouse experiment, putting
plants under three treatments: control (0% shade), 30% shade, and 70% shade. We monitored the
growth of individual ramets as well as clonal spread for 30 days. The number of new ramets (clonal
growth) increased exponentially in the three treatments, however this number was higher in shade
than in full sun plots. The size of floating leaves, submerged leaves, and rhizomes was higher
under shade treatments compared to individuals under sun treatment. Our results showed that S.
auriculata has a high growth performance under shade environments, including both clonal
growth and the size of individual ramets, similar to most land ferns. This species appears highly
plastic as it also thrives under bright sun, which appears to be a consistent pattern among fern
species in both terrestrial and aquatic environments. Because shade has been proposed as a
treatment to control and manage aquatic plants, application of this treatment should be carefully
considered depending of the species, especially when it involves aquatic fern species.

KEY WORDS.—Light, greenhouse, macrophytes, management, plasticity, shading net

Aquatic plants are important indicator species of ecological status in aquatic

ecosystems, but they can also cause harm especially when growth becomes
uncontrolled (Julien, Center, and Tipping, 2002; Lorenzi, 2000). These plants
can exhibit rapid growth rates and can influence aquatic environments, local

* Corresponding author. JESSICA CRISTINA CARVALHO MEDEIROS Tel.: þ55-11-3091-7600

Present address: Laboratory of Limnology, Bioscience Institute, Department of Ecology,
Universidade de São Paulo – USP, zip code 05508-090, São Paulo, SP, Brazil.
22 AMERICAN FERN JOURNAL: VOLUME 107, NUMBER 1 (2017)

economies, and human health (Jampeetong and Brix, 2009; Marcondes,

Mustafá, and Tanaka, 2003). Among the species of aquatic plants, ferns in
the genus Salvinia (Salviniaceae) are a potentially serious aquatic weed due its
clonal growth that can result in rapid expansion on the water surface (Coelho,
Lopes, and Sperber, 2005). Species of Salvinia may form thick mats covering
more than 100 km2 in just three months - choking lakes and waterways and
reducing populations of other aquatic plants and animals (Oliver, 1993). These
ferns can reproduce exponentially by vegetative fragments, with a leaf
doubling time of 3.5 days (Nichols, Couch, and Al-Hamdani, 2000). Salvinia
auriculata Aublet has been recognized as an invasive weed and an ecological
problem in many regions around the world (Room 1983, 1988, 1990).
Once these plants become invasive, it is essential to understand how they
perform under a variety of abiotic conditions. Important factors affecting
aquatic plants include light, depth, latitude, temperature, and water levels.
Among these factors, light, and in particular light intensity, is a very important
parameter because it influences photosynthesis and determines the ecological
distribution of plants (Hudon, Lalonde, and Gagnon, 2000; Larcher, 2003;
Poorter, 2001; Valladares and Niinemets, 2008; Zhu et al., 2014).
The effects of light availability on aquatic plants are of significant interest.
Macrophytes that live in full sun maximize light input and allocate resources
by increasing aboveground biomass, especially increasing leaf area and length
(Strand and Weisner, 2001; Vretare et al., 2001). Studies have shown that this
may enhance their photosynthetic area and increase growth (Garbey, Thiébaut,
and Muller, 2006). Other macrophyte species, such as the aquatic fern Marsilea
villosa Kaulf. grow best in moderate shade (Chau and Reyes, 2014). Indeed,
most pteridophytes grow best in shaded environments (Huang et al., 2011), but
the shade tolerance is species specific and depends on phase of life (Riaño and
Briones, 2015).
According to Sessa and Givnish (2014), light availability is the most important
variable driving morphological and physiological adaptation in land ferns.
However, the prevalence of this pattern in aquatic ferns is unknown. Due to
rapid dispersal rate, serious invasive potential, and limited information on the
impacts of light availability on aquatic ferns, the aim of this study was to test the
effects of different shade levels on growth of S. auriculata. Based on the premise
that light is an important variable driving morphological and physiological
adaptation in ferns, it was hypothesized that the ramets grow better under shade
conditions over time, and that full sun may reduce ramet growth.

MATERIALS AND METHODS

Studied species.—Salvinia auriculata is a free-floating fern found in diverse
habitats, such as rivers, reservoirs, and lakes, capable of growing and
developing very well in hot and sunny locations as in tropical regions
(Lorenzi, 2000). The plant consists of ramets connected by rhizomes, which
allows for the formation of broad colonies on the water surface. Each ramet
consists of a node with two floating green leaves and one submerged leaf that
MEDEIROS ET AL.: EFFECTS OF SHADE ON AN AQUATIC FERN GROWTH 23

functions as a root (Sculthorpe, 1967). The clone consists of genetically

identical ramets that grow through asexual reproduction and remain
connected, retaining integration between the parental ramet and its new
ramets (Charpentier, Mesléard, and Thompson, 1998).
The submerged leaf produces sori surrounded by a globose indusium, called
the sporocarp (De la Sota, 1962). The sporocarps are resistant structures and their
production occurs during the dry period, when clonal growth is unfavorable.
During flood periods, asexual reproduction is favored by formation of buds. Both
sexual and asexual reproductive strategies allow the species to survive in places
with seasonal inundation patterns, like temporary ponds that are subject to
drought and flood periods (Coelho, Lopes, and Sperber, 2005).
Plants and experimental design.—Ramets of Salvinia auriculata were
collected in a marginal pond that is influenced by the Rio Grande (218 11 0
15 00 S; 458 05 0 02 00 W), Minas Gerais, Brazil. In the greenhouse, the plants were
washed to remove the organic matter and soil particles. Efforts were made to
select ramets of the same size and appearance for the experiment.
From the selected plants, one ramet was placed in a 1L translucent plastic
pot. The pots contained 8 cm of Hoagland-Arnon solution (Hoagland and
Arnon, 1950) prepared in distilled water. The solution was exchanged once
per week in order to clean the pot and to avoid algal proliferation. We tested
the growth of individual ramets as well as clonal growth using three levels of
shade: control (under 100% of natural light in the greenhouse), 35% shade,
and 70% shade for a period of 30 days. To create the shade treatments we used
two different manufactured shading nets (35% and 70% shade) over the pots.
We used 15 pots per treatment, totaling 45 pots (N¼45).
The experiment was run for a period of 30 days, and every tenth day all the
ramets in the 45 pots were counted and measured. The initial time of the
experiment was considered time 0 (T0), 10 days after T0 (T1), 20 days after T0
(T2) and the last evaluation after 30 days (T3). We measured the: 1) number of
new ramets (clonal growth), 2) length and width of floating leaves, 3) length
and diameter of submerged leaf, 4) and length of rhizome. These
measurements can provide information about the growth of each plant
component, such as leaf expansion (floating and submerged leaves) and
rhizome expansion, which are responsible for the formation of the whole
clone, and the expansion of S. auriculata on the water surface. Clonal growth
was recorded as the number of new offshoots (ramets) produced.
Statistical analyses.—The data were normally distributed according to the
Kolmogorov-Smirnov test. To evaluate the treatment effect and temporal
variation of 1) the number of new ramets; 2) length and width of floating
leaves; 3) length and diameter of submerged leaf; 4) and rhizome length during
the study, we fitted a linear-mixed effects model. We specified random
intercepts per individual to account for the temporal pseudo replication in our
design. In the model, we considered treatment as a fixed factor with three
levels (control, 35% shade, and 70% shade); time was also considered as a
fixed factor and corresponded to days that we measured the variables in the
greenhouse (T0, T1, T2, and T3). In the linear-mixed effect models, we
24 AMERICAN FERN JOURNAL: VOLUME 107, NUMBER 1 (2017)

FIG. 1. Clonal growth of Salvinia auriculata under different shade treatments. The number of
ramets measured during the experiment in sun (control), 35% shade, and 70% shade in T0, T1 (10
days after begin the experiment), T2 (20 days) and T3 (30 days). Data indicate significant
differences among treatments at T3.

investigated differences between fixed factor levels (shade and time

treatments) using 95% confidence intervals estimated by the model. The
analyses were performed in R software.

RESULTS
Intact clones consisting of various sized ramets grew and filled the water
surface in all the pots. The number of new ramets (clonal growth) increased
exponentially in the three treatments (Fig. 1), however the number of ramets
was higher in both shade treatments (p,0.05) than in the control pots (0%
shade). The treatments alone did not have a significant effect on the number of
ramets (F¼0.41; p¼0.66). The number of new ramets increased with time
(F¼861.50; p¼0.001) and the interaction between treatments and time had a
significant effect on the number of ramets. Clonal growth at T3 was lower in
the control treatment and higher in the 35% and 70% shade (Fig. 1).
Considering the growth of individual ramets, the treatment alone did not
have an effect on the variables, but time had a significant effect; there was an
increase in all measurements (Table 1). The interaction between treatment and
time also had a significant effect in the measured variables (Table 1). The
length and width of floating leaves (Fig. 2a, b), length and diameter of
submerged leaf (Fig. 2c, d), and length of the rhizome (Fig. 2e), in T2 and T3,
were lower in control treatment (full sun) and higher in 35% and 70% shade.

DISCUSSION
Shade promoted an increase of individual ramet growth as well as clonal
growth in the aquatic fern Salvinia auriculata, supporting our hypothesis.
Under different levels of shade (35% and 70%), the plants produced longer
MEDEIROS ET AL.: EFFECTS OF SHADE ON AN AQUATIC FERN GROWTH 25

TABLE 1. Linear Mixed Model for the variables measured in the three treatments (control; 35%
shade; 70% shade) at four different times (T0 – equal conditions for all treatments, T1, T2 and T3).

Treatment Time Treatment x Time

Ramets measures (cm) F p F p F p
Length of floating leaves 0.68 0.50 949.83 ,0.001 3.47 0.03
Width of floating leaves 0.71 0.49 746.88 ,0.001 3.32 0.03
Length of submerged leaf 0.26 0.77 718.94 ,0.001 3.18 0.04
Diameter of submerged leaf 1.74 0.17 688.52 ,0.001 2.71 0.07
Rhizome length 1.83 0.16 788.44 ,0.001 3.51 0.03

rhizomes, larger floating leaves and submerged leaves, and a higher number of
new ramets (offshoots). The same pattern was observed in Marsilea villosa, an
endemic Hawaiian fern, which grows more vigorously under shade than under
sun, especially in moderate shade (Chau and Reyes, 2014), just as we observed
in S. auriculata.
Rapid growth is a feature of the life strategy of Salvinia auriculata. Species
with such a strategy quickly and effectively occupy free space (Seastedt, 2009).
For example, according to Galka and Szmeja (2013), the number of modules in
a S. natans clone increased at the rate of 0.6/day, initiating a new module
every second day on average. In our experiment, the colonies of S. auriculata
grew from a single ramet and covered the surface of the pots in just 30 days. In
addition, S. auriculata showed increased clonal growth under 35% shade, but
the new ramets also grew under the other treatments. Clone formation
undoubtedly is one of the features facilitating the expansion of S. natans, S.
molesta (Room, 1983), and S. auriculata (Coelho, Lopes, and Sperber, 2005),
especially under shade conditions, as we observed. Asexual modes are usually
induced by adverse environmental conditions or by a lack of resource
availability, inhibiting the normal life cycle (Barrett, Eckert, and Husband.,
1993). Clonal growth may enable to the plant to maximize the leaf
photosynthetic area, due to the numeric increase in physiologically indepen-
dent plant units (ramets) by clonal means. The production of new ramets
improves occupation and exploration of the environment. In addition, it
increases the probability that the genet will survive (Cook, 1979). In this way,
shade does not limit clonal growth of S. auriculata, on the contrary, it
produces more new ramets.
Our study shows that all measures of the individual ramet growth (length of
floating and submerged leaves, length of rhizome and diameter of submerged
leaf) were higher in moderate shade (35%) at the end of the experiment (T3)
(Fig. 2; Table 1). Thouvenot, Haury, and Thiébaut (2013) observed in the
aquatic plant Ludwigia grandiflora (Onagraceae) that stem lengths and leaf
areas were stimulated under reduced light (70% of sunlight intensity) and
were greater in the shade than in full sunlight. Zutshi and Vass (1971) also
reported considerable increase in the fresh weight of plants of S. natans under
partial shade in laboratory conditions. Macrophytes increase leaf area and
stem length in order to capture more light in the aquatic environment and to
26 AMERICAN FERN JOURNAL: VOLUME 107, NUMBER 1 (2017)

FIG. 2. The growth of individual ramets in Salvinia auriculata. Variables measured during the
experiment in sun (control), 35% shade, and 70% shade in T1 (10 days after initiating the
experiment), T2 (20 days), and T3 (30 days). (A) Length of floating leaves (cm); (B) Width of floating
leaves (cm); (C) Length of submerged leaves (cm); (D) Diameter of submerged leaves (cm); (E)
Rhizome length (cm). Different letters in each time indicate significant differences between
treatments (p.0.05).

optimize photosynthesis (Garbey, Thiébaut, and Muller, 2006). The higher

growth of S. auriculata under shaded conditions could be a compensatory
mechanism to acquire more light and maintain physiological function.
Over the duration of the experiment, we observed changes in how the
different treatments influenced both individual and clonal growth. At the
beginning of ramet development, all structures of the plant grew similarly in
both sun and shade. However, starting at T2 (20 days), whereas we observed
MEDEIROS ET AL.: EFFECTS OF SHADE ON AN AQUATIC FERN GROWTH 27

enhanced growth of the ramets and their structures in the shade, the full sun
treatment began to be detrimental for plant development. Although S.
auriculata can be found in environments with high incidence of sun, shade
environments could be the ideal conditions for this species development, as
has been observed in other fern species (Chau and Reyes, 2014).
Due to rapid growth, Salvinia can form a ‘‘mat’’ that covers large portions of
the water body where it grows (Gaudet, 1973; Nichols, Couch, and Al-
Hamdani, 2000; Room, 1990). Because S. auriculata can be an invasive weed,
control measures of this species (e.g. mechanical removal, biological and
chemical control) have been studied. Recently, Zhu et al. (2014) tested the
effect of shade on growth in the invasive macrophyte Hydrocharis morsus-
ranae (Hydrocharitaceae) and they reported that 70 and 100% shade was
effective at reducing biomass and controlling this species. Thus, in some cases
shading may be considered by aquatic plant managers as one of the possible
control methods (Zhu et al., 2014). Our findings indicate that 70% shade does
not prevent individual ramet growth or clonal growth of S. auriculata. Indeed,
the species grows better in moderate and intense shade (35% and 70%) than in
full sun.
To propose management tools for control of invasive and weedy species, it is
important to know their response to a variety of environmental conditions and
resources. In our study, S. auriculata responded differently than Hydrocharis
morsus-ranae did in the Zhu et al. (2014) study. Salvinia auriculata grows
better under shade environments, a pattern reported in some fern species from
both terrestrial and aquatic environments (Chau and Reyes, 2014; Huang et al.,
2011; Zutshi and Vass, 1971). It is evident that this plasticity in the growth of
individual ramets and the vigorous numerical increase of offshoots in the
clonal growth contribute to the ability of S. auriculata to thrive in a wide range
of habitats and explain its weedy behavior in some regions. Given that shade
has been proposed to control and manage aquatic plants, our study indicates
that it may not be recommended in all species, especially when it involves
aquatic ferns.

ACKNOWLEDGMENTS

The authors are grateful to Tamiris da Silveira Campos Resende for cooperating in the collections
and experiment, to Fabrı́cio José Pereira for his comments on the experiment elaboration, to editors
and anonymous reviewers for their contribution to the manuscript, and to Pró-Reitoria de Pesquisa
(PRP) for grant support.

LITERATURE CITED
BARRETT, S. C. H., C. G. ECKERT, and B. C. HUSBAND. 1993. Evolutionary processes in aquatic plant
populations. Aquatic Botany 44:105–145.
CHARPENTIER, A., F. MESLEARD, and J. D. THOMPSON. 1998. The effects of rhizome severing on the
clonal growth and clonal architecture of Scirpus maritimus. Oikos 83:107–116.
CHAU, M. M. and W. R. REYES. 2014. Effects of light, flooding, and weeding on experimental
restoration of an endangered Hawaiian Fern. Restoration Ecology 22(1):107–116.
28 AMERICAN FERN JOURNAL: VOLUME 107, NUMBER 1 (2017)

COELHO, F. F., F. S. LOPES, and C. F. SPERBER. 2005. Persistence strategy of Salvinia auriculata Aublet
in temporary ponds of Southern Pantanal, Brazil. Aquatic Botany 81:343–352.
COOK, R. E. 1979. Asexual Reproduction: A Further Consideration. The American Naturalist
113:769–772.
DE LA SOTA, E. R. 1962. Contributio al conocimento de las Salviniaceae neotropicales. II. Salvinia
auriculata Aublet. Darwiniana 12:499–513.
GALKA, A. and J. SZMEJA. 2013. Phenology of the aquatic fern Salvinia natans (L.) All. in the Vistula
Delta in the context of climate warming. Limnologica 43:100–105.
GARBEY, C., G. THIÉBAUT, and S. MULLER. 2006. An experimental study of the plastic responses of
Ranunculus peltatus Schrank to four environmental parameters. Hydrobiologia 570:41–46.
GAUTED, J. J. 1973. Growth of a floating aquatic weed, Salvinia, under standard conditions.
Hydrobiologia 41:77–106.
HOAGLAND, D. R. and D. I. ARNON. 1950. The water-culture method for growing plants without soil.
Berkeley, California Agricultural Experiment Station.
HUANG, D., L. WU, J. R. CHEN, and L. DONG. 2011. Morphological plasticity, photosynthesis and
chlorophyll fluorescence of Athyrium pachyphlebium at different shade levels.
Photosynthetica 49:611–618.
HUDON, C., S. LALONDE, and P. GAGNON. 2000. Ranking the effects of site exposure, plant growth form,
water depth, and transparency on aquatic plant biomass. Canadian Journal of Fisheries and
Aquatic Sciences 57:31–42.
JAMPEETONG, A. and H. BRIX. 2009. Nitrogen nutrition of Salvinia natans: Effects of inorganic
nitrogen form of growth, morphology, nitrate reductase activity and uptake kinetics of
ammonium and nitrate. Aquatic Botany, 90:67–73.
JULIEN, M. H., CENTER,T. D. and P. W. TIPPING. 2002. Floating Fern (Salvinia). In Biological control of
invasive plants in the eastern United States. USDA Forest Service, Publication 4 17–32.
LARCHER, W. 2003. Physiological Plant Ecology. Ecophysiology and Stress Physiology of Functional
Groups, 4th edition, Springer, New York, New York, USA.
LORENZI, H. 2000. Plantas Daninhas do Brasil. 3. ed. Nova Odessa: Instituto Plantarum.
MARCONDES, D. A. S., A. L. MUSTAFÁ and R. H. TANAKA. 2003. Estudos para manejo integrado de
plantas aquáticas no reservatório de Jupiá. In: THOMAZ, S. M.; BINI, L. M. Ecologia e manejo
de macrófitas aquáticas. Maringá EDUEM. 299–317.
NICHOLS, P. B, J. D. COUCH, and S. H. AL-HAMDANI. 2000. Selected physiological responses of Salvinia
minima to different chromium concentrations. Aquatic Botany 68:313–319.
OLIVER, J. D. 1993. A Review of the Biology of Giant Salvinia (Salvinia molesta Mitchell). Journal of
Aquatic Plant Management 31:227–231.
POORTER, L. 2001. Light-dependent changes in biomass allocation and their importance for growth
of rain forest tree species. Functional Ecology 15:113–123.
RIAÑO, K. and O. BRIONES. 2015. Sensitivity of three tree ferns during their first phase of life to the
variation of solar radiation and water availability in a Mexican cloud forest. American Journal
of Botany 102:1472–1481.
ROOM, P. M. 1983. ‘Falling apart’ as a life style: the rhizome architecture and population growth of
Salvinia molesta. Journal of Ecology 71:349–365.
ROOM, P. M. 1988. Effects of temperature, nutrients and a beetle on branch architecture of the
floating weed Salvinia molesta and simulations of biological control. Journal of Ecology
76:826–848.
ROOM, P. M. 1990. Ecology of a simple plant-herbivore system: biological control of Salvinia.
Trends in Ecology & Evolution 5:74–79.
SCULTHORPE, C. D. 1967. The Biology of Aquatic Vascular Plants. London: Edward Arnold.
SEASTEDT, T. 2009. Ecology: Traits of plant invaders. Nature 459:783–784.
SESSA, E. B. and T. J. GIVNISH. 2014. Leaf form and photosynthetic physiology of Dryopteris species
distributed along light gradients in eastern North America. Functional Ecology 1:108–123.
STRAND, J. A. and S. E. B. WEISNER. 2001. Morphological plastic responses to water depth and wave
exposure in an aquatic plant (Myriophyllum spicatum). Journal of Ecology 89:166–175.
THOUVENOT, L., J. HAURY, and G. THIÉBAUT. 2013. Seasonal plasticity of Ludwigia grandiflora under
light and water depth gradients: An outdoor mesocosm experiment. Flora 208:430–437.
MEDEIROS ET AL.: EFFECTS OF SHADE ON AN AQUATIC FERN GROWTH 29

VALLADARES, F., and U. NINEMETS. 2008. Shade tolerance, a key plant feature of complex nature and
consequences. Annual Review of Ecology, Evolution and Systematics 39:237–257.
VRETARE, V., S. E. B. WEISNER, J. A. STRAND, and W. GRANÉLI. 2001. Phenotypic plasticity in
Phragmites australis as a functional response to water depth. Aquatic Botany 69:127–145.
ZHU, B., M. S. ELLIS, K. L. FANCHER, and L. G. RUDSTAM. 2014. Shading as a Control Method for
Invasive European Frogbit (Hydrocharis morsus-ranae L.). Plos One 9:1–6.
ZUTSHI, D. P. and K. K. VASS. 1971. Ecology and production of Salvinia natans Hoffim. in Kashmir.
Hydrobiologia 38:303–320.