Abstract
- Silicon mediated plant–herbivore interactions have gained increasing
recognition and have now been studied in a wide range of species. Many
studies have also considered accumulation of Si by plants as a process
largely driven by geo-hydrological cycles.
- To identify factors driving the water - plant Si - herbivore nexus we
analysed the concentration of Si in fibrous tussock sedge (Carex
appropinquata ), the population density of the root vole
(Microtus oeconomus ) and the ground water level, over 11 years.
- The largest influence of autumn Si concentration in leaves
(Sileaf) was the level of the current year’s ground
water table, which accounted for 13.3% of its variance. The previous
year’s vole population density was weakly positively correlated with
Sileaf and alone explained 9.5% of its variance.
- The only variable found to have a positive, significant effect on
autumn Si concentration in rhizomes (Sirhiz) was the
current year spring water level explaining as much as 60.9% of its
variance.
- We conclude that the changes in Si concentration in fibrous tussock
sedge are predominantly driven by hydrology, with vole population
dynamics being secondary. Our results provide only partial support for
the existence of plant-herbivore interactions, as we did not detect
the significant effects of Si tussock concentration on the vole
density dynamics. This was mainly due to low level of silification of
sedges, which was insufficient to impinge herbivores. Future studies
on plant–herbivore interactions should therefore mainly focus on
identification of mechanisms and conditions allowing plants to
accumulate silica at the levels sufficient to act as an anti-herbivore
protection.
Key-words : ground water level, plant defense, population
density, tussock sedges, voles
1 INTRODUCTION
Recent studies suggest that the dynamics of the small mammal population
are shaped primarily by the external factors (so-called: trophic
interactions) such as predation and availability or quality of food
(Klemola, et al. 2000; Lambin, et al., 2000; Oli, 2019, but see
Adreassen et al., 2013). Among factors related to food quality, root and
leaf silication induced by past overgrazing has recently received
particular attention. A number of studies demonstrated that abrasive
properties of silicon contained in the plant tissues deteriorate
herbivore teeth (Calandra et al., 2016), cause abrasion of intestinal
villi (Wieczorek et al. 2015b) as well as a reduction in body mass and
survival prospects (Zub et al., 2014; Wieczorek et al., 2015a).
Furthermore, plant responses to herbivory pressure has been observed
within laboratory based studies, in which pressure from field voles
(Microtus agrestis ) increased the presence of silicon in grass
tissues by 400% (Massey & Hartley, 2006).
Yet, the results of the field studies on the associations between
induced silicon plant defence and mammalian herbivory have not been as
clear as the laboratory ones. Studies conducted in Norway have shown
that the induced grass response to the herbivory manifested by an
increase in the content of silicon in plant tissues is extremely
variable and depends, not only on the pressure of herbivorous mammals
(rodents and reindeer), but also on the location, plant species and its
genotype (Soninen et al., 2012). Quigley et al. (2020) demonstrated that
Si concentration in grass leaves did not respond to large mammalian
grazer exclusion studied in a climatic gradient, but was strongly
affected by nutrient availability. In turn, field experiments carried
out in Kielder Forest (UK) showed that after several months of density
manipulation the level of silicon in wavy hair grass (Deschampsia
caespitosa ) leaves decreased by 22% on sites where field vole density
had been reduced, but the increase in silicon content did not affect
body weight of voles, nor their spring population growth rate or
survival, which suggests that plant quality hypothesis is unlikely to
explain the observed cyclicity in the Kielder Forest field vole
population (Ruffino et al., 2018). Likewise, (Wieczorek et al., 2015a;
but see Soninen et al., 2017) showed that vole herbivory elevated
silicon levels in sedges, albeit with no detectable effect on the winter
survival rates of voles.
The above inconsistencies may simply stem from the lack of sufficient
statistical power of these analyses, since the longest of them lasted
between three (Soninen et al., 2012) to four years (Wieczorek et al.,
2015a) and are therefore based on a small number of degrees of freedom.
Long-term studies, able to capture the time-course of the putative
plant-herbivore association, are particularly needed because changes in
silicon levels in plant tissues are not only due to grazing, but are
also responsive to abiotic factors, chiefly water availability, which
drives silicon absorption in form of silicic acid (Raven, 1983;
Sangster, Hodson, & Tubb, 2001; Kindomihou, Sinsin & Meerts, 2006;
Faisal et al., 2012; Brightly et al,, 2020). The effect of the water on
the induction of silicon in sedge leaves has been indirectly
demonstrated in European (Wieczorek et al., 2015a) and in African
ecosystems (Quigley & Anderson, 2014).
To address such short-term limitations in previous research, we analysed
an 11-year time series of: ground water level; population dynamics of
the root vole (Microtus oeconomus ); and silicon levels in the
tissues of the fibrous tussock sedges (Carex appropinquata ,
Schumacher, 1801) – the main food source of the voles. To our
knowledge, this is the longest time series ever used to test the effect
of plant defences and water availability in plant-herbivore
interactions. We tested whether: sedges induce silicon defences in
response to the grazing by root voles; the feedback of silicon in sedges
influence vole population dynamics; and whether ground water level
influenced the plant-herbivore system. Following Wieczorek et al.,
(2015a) we did so by taking into consideration Si concentration in
rhizomes (Sirhiz) as well as in leaves
(Sileaf), as the dynamics of the plant-herbivore
interaction can be different depending on the part of the plant. We
predicted that Sileaf should be positively affected by
the previous year’s vole population density. However,
Sirhiz should be primarily stimulated by the same year
herbivore-incurred damage. As Si uptake by sedges is positively driven
by water availability, we also surmised that Sileaf and
Sirhiz were likely to be positively affected by the
level of ground water table in previous and/or current spring.
Conversely, year-to-year changes of vole population density should be
inversely correlated with the Sileaf and
Sirhiz with a one-year time lag.
2 STUDY AREA
The study was conducted in the Lower Basin of the Biebrza National Park,
NE Poland (53°36′18″N, 22°55′36″E). The study area is located in a
homogenous sedge wetland with vegetation dominated by plants fromCyperaceae family. The main plant species in the Park is the
fibrous tussock sedge, which covers 85% of the area and forms
hummock-hollow structures (Matuszkiewicz, 2020). The wetland has a
seasonal water regime with the highest level during spring, when
flooding is frequent. The climate is characterized by long winters
(>100 days), short and early springs, and short summers
(77-85 days).
The main herbivores in the area are rodents and moose (Alces
alces ). Root voles are the dominant rodent species in this habitat,
making up 90% of small mammal communities (Borowski, 2002; 2011). We
worked with a natural population of root voles which displays cyclical
dynamics (Borowski, 2011). The study began in 2007, during which time
various vole population peaks (2008–2009, 2015-2016) and crashes (2007,
2017) occurred (Fig. 1).
2.1 VOLE DENSITIES
In order to estimate vole population sizes we carried out
Capture-Mark-Recapture (CMR) trapping free-living populations in two
sites, each 0,6 ha large and separated by 3 km. The first site, called
Gugny was trapped between 2007 and 2019, while the second, called
Barwik, was trapped for 2012 to 2019. Trapping occurred at each site
once a year in autumn (November).
Vole population size estimates were converted to density per ha based on
a CMR method (see Borowski, 2011 for details).
2.2 SEDGE SAMPLING
Sedges are the main food of root voles, both in summer and winter. In
summer, the diet is dominated by green parts of the plant, though in
late autumn and winter voles also eat the woody parts, such as dry
rhizomes and roots (Tast, 1966; Gębczyńska, 1970; Batzli & Henttonen,
1990).
In the Barwik and Gugny sites, every November from 2014 to 2019, we
collected 10 haphazardly selected samples of sedge tussocks at each
site. This resulted in 120 Carex tussocks samples (10 per year in
each site over 6 years). To determine for Si content, from each tussock
we took biomass sample composed of leaves or rhizomes produced in the
present year, physically connected with the tussocks (representing a
single plant). Dead parts of the plant or decaying litter were
discarded. As voles do not feed on decaying litter, we selected only
leaves and rhizomes which were both physically connected and also
composed of dried, non-decomposing tissues. We separated leaves from
rhizomes, and samples were cleaned under running water, dried at 80 °C
to a constant mass and stored in separate plastic bags for further
analysis.
Data on Si concentrations in leaves and rhizomes from Gugny site (from
2007-2011) were collected in a similar manner as described above, for
details see Wieczorek et al., (2015a). We calculated mean value of Si
concentration in leaves or rhizomes from 10 samples collected for each
year and site, thus our sample size was N = 17 (5 samples for period
2007-2011 from Gugny and 12 samples for period 2014-2019 from Gugny and
Barwik).
2.3 WATER LEVEL
We measured water level using piezometers, with the instruments in both
study sites, each located ca. 5 km from the Biebrza River. Five to six
measurements of water level (m) were taken in May and June using the
same respective piezometer. These measurements were then averaged to be
used later used in analysis. The river and its floodplain form an
interconnected spatially distributed system (Fisher et al.,1998) which
experiences regular flooding, which occurs in spring.
2.4 CHEMICAL ANALYSIS
The above ground biomass and roots were separated and milled using a
Tecator Cyclotec1093 mill. Each 150 mg sample of biomass was then
digested in a 9:1 mixture of concentrated HNO3 and HF in
Speedwave Four apparatus (Berghof, Germany), with temperatures reaching
a maximum of 230 oC. Si content in digested material
was measured using atomic an absorption spectrometer, Contraa700
(Analytik Jena, Germany), in nitrous oxide-acetylene flame with 251.6 nm
wavelength. Recovery of Si was determined using NCS DC 73349 certified
material (recovery was within 91% to 102% with a mean of 96%).
2.5 STATISTICAL ANALYSES
To analyse the data we used Generalized Linear Mixed Models (GLMM) withlog link function. We log-transformed all, but one (water level),
variables to correct for their right skewed distribution. Model
assumptions were checked using residual plots. These confirmed: ε was
normally distributed, model fits lacked heteroscedasticity, and no
observations were disproportionately influential in any of the models.
To identify factors affecting the November Si concentrations in leaves
or rhizomes we used the following variables, with their respective
interactions, as the fixed terms: the autumn density of voles in the
previous (t -1) and the current (t ) year, the spring water
level from the present (year t ), and Si concentration in leaves
or rhizomes in the present year (year t ). In the final models
only the significant interaction terms were retained. The study site
(Gugny or Barwik) and year of study were used as a random factors. As
the “study site” random factor has only two levels in some models it
caused singularity and then was removed. We used year as a random factor
to resolve the problem of autocorrelation of Si concentration between
year t and t -1.
We used similarly structures GLMM model with log link function to
analyse vole density in year n. The model included: Si concentration in
the leaves and rhizomes (in year t ); the previous year’s vole
density (year t-1 ); the current (year t ) water level as
fixed effects. As with the Si models, site and year were included
as a varying intercept random effect.
For all models we calculated the R-squared values as marginal and
conditional R2 statistics (according to Nakagawa et
al., 2017). The marginal R2 considers only the
variance of the fixed effects, while the conditional
R2 takes both the fixed and random effects into
account. We also provided values of part (semi-partial)
R2 as the metrics of variance explained uniquely by a
particular predictor.
All statistical analyses were made using packages lmerTest (Kuznetsova
et al., 2017), sjPlot (Lüdecke, 2020) and partR2 (Stoffel et al.,2020)
in the R software.
3 RESULTS
Preliminary analyses revealed that Si concentration in sedges, vole
density and ground water level varied significantly between years of
study (p < 0.001 for each of the three variables, Table 1,
Figure 1).
GLMM revealed that autumn Sileaf was positively affected
by the ground water level in the spring of the same year (Table 1). Same
analysis also revealed the effect of the population density of voles
recorded in the previous autumn (year t -1, Table 1, Figure 2).
Although this effect did not reach statistical significance, it
nevertheless explained 9.5% out of 22.6% of Sileafvariation accounted for by all fixed factors (Table 1).
Concentration of Si in rhizomes (Sirhiz) was
significantly affected only by the ground water level in spring, whereas
the effect of vole density in the same year (t ) and previous year
(t -1) was weak and not significant. All fixed effects explained
77.7% of Sirhiz variation (Table 2). When the spring
water level was removed from the model, all remaining fixed effects
explained only 16.9% of Sirhiz variation and density of
voles explained alone 16.1% of Sirhiz variation. In the
resulting model the density of voles in year t -1 became
marginally significant (GLMM, coefficient estimate = 0.12, CI: 0.00 –
0.25, p = 0.053).
Neither Sirhiz, Sileaf and vole density
in year t -1 nor water level in current year, significantly
affected the density of the vole population in the current year
(t ) (Table. 3).
4 DISCUSSION
Our long term study revealed that (1) the amount of Si in leaves was
positively related to current year water level in spring and, to a
smaller extent, vole population densities from the previous year; (2)
spring ground water level had a strong and positive influence on rhizome
Si content, but (3) neither Si concentration in leaves or rhizomes, or
water level affected the root vole population density. Thus, our
findings partly corroborated the results of an earlier study by
Wieczorek et al., (2015a) carried out on the same field study system.
Above all, however, our results demonstrate that the
plant-herbivore-water-regime nexus is more complex than has been
described from laboratory (Seldal et al., 1994) and enclosure
experiments (Agrell et al., 1995).
Sileaf saw notable increases in years following
especially high root vole densities (> 200 individuals per
hectare in 2008, 2014 and 2015, Fig. 1). Such a one- year- delayed plant
response to grazing is in agreement with the predictions of the Plant
Defence Hypothesis (Haukioja, 1980; Underwood, 1999) and suggests that,
in those particular year’s, herbivore densities were high enough to
elicit a silification defence. Our study follows Wieczorek et al.,
(2015a), who similarly demonstrated that the high pressure from
free-living small herbivores may elevate bio-mineralisation of tissues
of grasses under natural conditions. This corroboration is important,
because the findings presented in Wieczorek et al., (2015a) have been
questioned by Soininen et al., (2017) on statistical grounds.
A similar mechanism of silification of grasses caused by vole grazing
was observed in studies conducted both in a laboratory (Reynolds et al.,
2012) and also in a landscape scale experiments (Ruffino et al., 2018).
The question therefore arises of why so few confirmations of
silica-induced defence mechanisms in grasses generated by the
herbivorous mammals are detected in the wild, while it is so readily
detected in laboratory experiments? The most intuitive explanation is
that the diet of wild herbivores in natural grasslands is much more
diverse than in the laboratory studies, which results in insufficient
grazing pressure to induce defence mechanisms in a given plant species.
Fortunately, in our study system in the Biebrza National Park,
homogenous meadows consist almost exclusively of the one Carex species
– the tussock sedge, constituting a primary food source of voles
(Gębczyńska, 1970). Therefore, this simplest possible one plant- one
vertebrate herbivore system is best suited for testing the existence of
induction of silicon deposition as a defence mechanism against grazing.
The second possibility is that the elevation in Si concentration in
plants is most detectable at high densities of herbivorous mammals which
is easily replicated within laboratory settings (e.g. Massey & Hartley,
2006) but difficult to capture in natural ecosystems. This may explain
why the field studies conducted by Soninen et al., (2012) and Quigley et
al. (2020) did not reveal consistent relationships between plant silicon
concentrations and grazing. The results of our study and other two field
experiments (Wieczorek et al., 2015a; Ruffino et al., 2018) indicate
that such relationships are only detectable following response of plants
to especially high herbivore densities, in this study above 200
individual/ha.
The key factor, however, complicating the analyses of the
plant-herbivore interaction in our study system is the ground water
level. The uptake and deposition of silicon in wetland plants is driven
by hydrological and climatic factors (Struyf & Conley, 2009; Struyf et
al., 2010; Schoelynck et al., 2014), because silicic acid uptake in
grasses is largely passive and determined by transpiration rate
(Sangster, Hodson, & Tubb, 2001, but see Quigley et al. 2020).
Wieczorek et al., (2015a) found that ground water level positively
affects Sirhiz. This result has been questioned by
Soninen et al. (2017) who asserted that the effect of the ground water
level in Wieczorek et al.’s, (2015a) study cannot be statistically
separated from that of the vole density. Our present analysis, carried
out on a much larger data set, allowing for an effective statistical
control of the collinearity between independent factors, did support the
existence of a strong positive effect of the ground water level on Si
accumulation in leaves and rhizomes (Table 1 and 3 respectively).
However, we found no evidence that Sirhiz and
Sileaf, in the same year, are correlated, while
Sirhiz in consecutive years was positively correlated.
Thus, the dynamics of Si deposition in leaves and rhizomes follow
different paths, although in both plant parts it is driven by the
prevailing water regimen (Table 1 and 2).
Although high grazing pressure of voles elevated Si level in sedge’s
leaves in an apparent delayed density-dependent manner, it did not
affect vole population densities between years. In agreement with this
finding, Wieczorek et al., (2015a) found that the winter survival of
voles was not associated with vole faecal Si concentration. In
principle, this concentration should be correlated with
Sirhiz, because the Sirhiz is correlated
between subsequent years of study, and thus, should also faithfully
reflect winter Si concertation in rhizomes being the food base of
overwintering voles. The lack of the effect of silification of sedges on
the vole population dynamics was likely due to low
Sileaf and Sirhiz, which in most years
of our study remained at the level of less than 1% of dry mass (Fig.
1). This level was 3-6 times lower than that reported in leaves ofDeschampsia caespitosa by Massey et al. (2008)- a study
demonstrating negative effect of plant silification on the population
growth and individual performance of voles (Microtus agrestis ).
Likewise, Wieczorek et al.’s (2015b) study demonstrating the abrasive
effect of silica on intestinal villi of voles used sedge-based diet
containing 1.87% of Si in dry mass— a concentration higher than that,
reported here.
5 CONCLUSIONS
In conclusion, our results indicate that silification process of
rhizomes of sedges in our study area is mainly driven by the
hydrological cycles. Si concentration in leaves appears to be dependent
of the ground water level, and slightly positively affected by the
previous year’s vole population density. However, this effect does not
create a feedback loop predicted by the Plant Defence Hypothesis, as
silification of sedges was insufficient to negatively affect the root
vole demography. Therefore, future research carried out on the on the
Plant Defence Hypothesis should mainly focus on clarifying why and under
what circumstances plants are able to accumulate silica at the levels
sufficient to impinge herbivores, thereby triggering plant-herbivore
interactions.
ACKNOWLEDGEMENTS
We are grateful to the Institute of Biology in Białystok for allowing us
to use their Field Station in Gugny. Deon Roos provided valuable
comments and linguistic correction on earlier draft of this paper.
Financial support was provided by the Polish National Science Centre
(grant 2011/01/B/NZ8/04259 to ZB) and the Forest Research Institute
(grant 900-418 to ZB). This work complies with the cur-rent laws of
Poland.
AUTHORS ’ CONTRIBUTIONS
Z.B., K.Z. and M.K. conceived the ideas and designed methodology; Z.B.
and K.Z. collected the data; K.Z., M.K. and Z.B. analysed the data, M.S.
and M.S-M. performed chemical analyses; Z.B., M.K. and K.Z. led the
writing of the manuscript. All authors contributed critically to the
drafts and gave final approval for publication.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
DATA AVAILABILITY STATEMENT
Data will be available at the Dryad Digital Repository
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