In of June 2008, multiple individuals of *T. triandra* and *D. macroblephara*, were collected from four grassland sites spanning the ecosystem's soil and rainfall gradients and transplanted into a common garden constructed at the Serengeti Wildlife Research Centre (SWRC; **Figure 1**). *D. macroblephara* was collected from Barafu (BRS) in the southeastern plains, *T. triandra* was collected from Klein's camp (KCW) in the northern corridor, and both species were collected from the Togoro plains (TOG) and Musabi plains (MSB). Grasses were planted in three replicate blocks; each block consisted of a 6 × 9 m fenced area cleared of all vegetation and included six 2 × 2 m equally spaced plots. Three randomly selected individuals of *T. triandra* and *D. macroblephara* (one from each collection site) were planted in each plot. Mean ± SE tiller number of initial *D. macroblephara* individuals was 13.59 ± 0.67, and *T. triandra* transplants had a mean ± SD = 9.08 ± 0.74 tillers. Plots were randomly assigned to one of the six combinations of clipping (two levels: clipped and unclipped) and watering (three levels: high, ambient and low). Water was manipulated by diverting rainfall with five 25 cm strips of clear plastic roof material placed across the plots at ∼6 cm height and slanted slightly to promote water runoff; the treatments were designed to intercept approximately 50% of ambient rainfall. Grasses were planted between the roof strips and at least 45 cm from the nearest grass to provide room for growth and to prevent light competition. To control for any potential effects of the roofing material, all plots were overlaid by the roofing material but holes were drilled in the high and ambient treatments so that no water was diverted and all rainwater reached the plot. Rainfall was collected, and the high water treatment was augmented with an amount that totaled ∼150% of ambient every 2 weeks. Rainfall at SWRC is approximately 700 mm yr−1 and the high water treatment was augmented to approximately 1000 mm yr−1. Soil moisture at a depth of 12 cm was estimated via time-domain reflectometry monthly in each plot with a FieldScout TDR 100 (Spectrum Inc.; Aurora, IL, USA). In the clipping treatment, plants were defoliated to approximately 50% of their aboveground biomass every 2 months. Clipped biomass was dried, weighed, and summed with harvested biomass to arrive at a final biomass for clipped plants. Unclipped plants were not defoliated throughout the entire duration of the experiment. Plants were grown in the common garden for 10 months, until they were harvested in April 2009. All plants still alive at the harvest were separated into above- and belowground fractions; the aboveground fraction was further separated into leaf and stem. All plant material was dried and weighed in the lab at SWRC; leaf fractions were transported to the US, ground in a Cyclone sample mill (UDY corp.; Fort Collins, CO, USA) and stored until they were analyzed for leaf silica at Wake Forest University.

#### *Biogenic silica quantification*

Silicon content of leaf tissue was quantified by plasma spectroscopy (ICP-OES) after autoclave-induced digestion (AID) following the methods of Kraska and Breitenbeck (2010; modified from Elliott and Snyder, 1991). Briefly, 100 mg of dried, ground sample was wetted with 1-octanol in a 50 ml vortex tube. Next, 2 mL of 50% H2O2 and 3.5 mL of 50% NaOH were added. Samples were vortexed several times, until the reaction ceased, and autoclaved loosely capped at 121◦C (20 psi) for one hour. Deionized water was added to 50 mL. Samples were brought to acidic pH using concentrated HCl, and diluted 1:10 in deionized water before analyzing by ICP-OES. Si content of samples was calculated by fitting peak intensity at 251 nm to a standard curve (0.1 –10 ppm Si; *r*2 ≥ 0.998). The standard curve was validated with a reference material of *Schizachyrium scoparium* (14 g/kg Si). Si values were converted to silica (SiO2) content by dividing by a conversion factor of 0.4674, since this more commonly reported value represents biogenic silica (phytolith) content of plant tissue.

#### *Statistical analysis*

A major goal of our statistical analysis was to understand how defoliation, water addition and their interaction, influenced leaf silica concentration in these two dominant Serengeti grasses. Therefore, we used linear mixed effect (LME) models to test the effects of species, clipping, and water on foliar silica accumulation. The model included species, clipping, and water as fixed factors, while block was included as a random effect to account for the spatial design. Analysis was performed using the lmer function in *lme4* package of R statistical environment version 2.11.1 (Bates et al., 2014; R Development Core Team, 2014), with the original model structure: lmer(SiO2 ∼ species ∗ clip ∗ water + (1| block)), where "species∗clip∗water" represents the three-way fixed interaction effect and "(1| block)" represents the random intercepts that are estimated for each block. The function "step" from the R package *lmerTest* was used to simplify the model so that only significant fixed effects remained in the model, and pairwise contrasts for individual treatment effects were subsequently calculated using the "ghlt" command and a Tukey's test from the R package *multcomp* (Hothorn et al., 2008).

A second model was employed to explore the extent to which collection site could explain variation in foliar silica content for both species. Because we did not have sufficient replication of individual grasses at the site level, we could not explore the effects of site in a full model crossed with our other treatments. Consequently, we used a reduced statistical model to explore the main effects of site on leaf silica for each individual species averaged over all levels of water and defoliation. The model we employed for each species separately was: lmer(SiO2 ∼ site + (1| block)). Model simplification was again conducted via the "step" function in R.

Finally, soil moisture was statistically compared across treatments using a model with water as a fixed effect and time and block as random effects using the lmer function in *lme4* package of R as described above; the "ghlt" command from the R package *multcomp* was used to conduct a Tukey's *post hoc* comparison of means.

#### **LITERATURE REVIEW**

In order to identify general trends in plant response to defoliation, we conducted an extensive search of primary literature sources to identify studies which provided data on silica content of Poales under both control and defoliation treatments. We used Google Scholar and Web of Science to identify appropriate primary research articles by using combinations of search terms such as "clipping,""grazing,""defoliation,""silica," and "phytolith." Within the order Poales, defoliation studies were only identified for grasses, so the final literature review is limited to Poaceae. We restricted our search to studies containing species-specific silica values in order to avoid the potential confounding effects of differing community composition on bulk, plot level silica content. These studies included laboratory studies and field studies in which grazing was experimentally prevented by herbivore exclosures. Grazing studies encompassed insect, small mammal, and large mammal herbivory, and the intensity of defoliation varied within and among studies (see Discussion). Species-specific Si values were converted to SiO2 when necessary, assigned a unique identifier, and plotted as the log ratio normalized difference between defoliated and non-defoliated plants at the species level. Studies were then organized by defoliation method and grazer type and assigned to a general defoliation response category to facilitate interpretation of the silica response. Those cases with a ≥ 20% increase in silica following defoliation were assigned a "+," those which decreased ≥20% were assigned a "−," and those exhibiting less than a 20% relative change in silica content were assigned a "0."

# **RESULTS**

#### **SERENGETI COMMON GARDEN**

Mean ± SE soil moisture values were 11.1 ± 1.6% for the high, 9.8 ± 1.5% for the ambient and 9.7 ± 1.5% for the low watering treatment. *Post hoc* statistical comparisons with a Tukey's test demonstrated that the low and ambient water treatments were not statistically different from one another (difference = −0.1, *z* = −0.26, *p* = 0.96), but that the high water treatment was statistically greater than the low (difference = 1.4,*z* = −2.9, *p* = 0.007) and ambient (difference = 1.3, *z* = 2.73, *p* = 0.01) treatments. Therefore, the ambient and low water treatments are combined for the remainder of the manuscript (referred to as "ambient" from here onward) and compared to the high water treatment in all analyses of watering effects.

Both the bunch-grass *T. triandra* and lawn-grass *D. macroblephara* were relatively high Si-accumulators: *T. triandra* had a mean ± SE foliar SiO2 content of 3.7 ± 0.25 % dw (*n* = 20), while *D. macroblephara* had a mean ± SE SiO2 of 2.7 ± 0.10 % (*n* = 41). Neither species showed a significant response to clipping as a main effect (*p* = 0.976) or as an interaction effect when clipping was crossed with watering level (*p* = 0.453; **Figure 2**). However, there was a significant species by watering interaction effect (*p*<0.05, Supplementary **Table S1**) indicating that leaf silica concentrations of the two species differed in response to the watering treatment. This result arose because individuals of *T. triandra* that were watered had a higher leaf silica concentration (4.4 ± 0.3%) compared to those *T. triandra* plants that were maintained at ambient soil moisture levels (3.3 ± 0.3%). In contrast, no

significant change in SiO2 content was observed between watering treatments for *D. macroblephara* (**Figure 2**).

In the second analysis, in which the statistical model included the main effects of plant collection site for each species separately, *D. macroblephara* exhibited considerable within-site variability and consequently no statistically significant differences across sites. In contrast, the SiO2 content of *T. triandra* plants varied significantly among sites (**Figure 3**; see **Figure 1** for sites): Silica was lowest for plants collected from TOG (mean ± SE = 2.67 ± 0.63%), intermediate for plants from MSB (mean ± SE = 3.97 ± 0.55%), and highest for plants from KCW (mean ± SE = 4.18 ± 0.53%). Independent contrasts indicated that grasses from the low rainfall site TOG

**FIGURE 3 | Foliar silica content of each species in relation to plant collection sites.** Both species were collected at the intermediate sites, MSB and TOG. Themeda (TheTri) does not occur at the shortgrass plains site BRS, and Digitaria (DigMac) does not occur at KCW in the northern woodlands. Sites are arranged in order of increasing mean annual precipitation: BRS = 498 mm/yr, TOG = 676 mm/yr, KCW = 766 mm/yr, MSB = 891 mm/yr.

accumulated significantly less silica than those collected from MSB (difference = −1.30, *z* = −2.822, *p* = 0.005) and KCW (difference = −1.51, *z* = −2.408, *p* = 0.016).

#### **LITERATURE REVIEW**

We identified 11 studies conducted from 1974-present which recorded species-specific silica induction under control and defoliation treatments. These studies represented 34 "cases" of potential silica induction for 15 different grass species (Supplementary **Table S2**). Due to the limited number of studies conducted and lack of species-specific replication, we could not employ meta-analytical statistical approaches; instead, general responses were considered and interpreted. Eleven of fifteen species surveyed are C3, and all are perennials, except for *Poa annua*. Silica content of surveyed species ranged from <0.5% dry weight (nondefoliated *Anthoxanthum odoratum*) to >7% (grazed *Pascopyrum smithii*, *Deschampsia cespitosa,* and *Eustachys paspaloides*). To avoid pseudoreplication, we reported silica values for seasonal or site-level maxima for the three studies which reported multiple species-specific responses to the same defoliation method (Brizuela and Detling, 1986; Cid et al., 1990; Banuelos and Obeso, 2000). The majority of studies surveyed suggest that plants are able to respond to defoliation by altering their silica uptake, and indeed we observed an overall trend of silica induction following defoliation (**Figure 4**). Despite this general trend, several studies found no significant change in silica content of defoliated grasses, defined here as less than a 20% change in foliar silica content in either direction. The literature review revealed substantial variation in the silica responses of the different species studied. For example, the C3 grass *Festuca* increased silica content more than 350% in response to herbivory by voles (Massey et al., 2007), while silica content of *Pascopyrum* decreased by approximately 50% following clipping (Cid et al., 1990). In general, the magnitude of induced silica uptake was greater under natural defoliation (i.e., grazing) than for mechanical clipping. Further, the magnitude of silica increase when defoliation stimulated uptake

much greater than the decrease in silica levels observed in the two studies in which clipping triggered down regulation of silica levels.

#### **DISCUSSION**

#### **VARIATION IN SILICA BETWEEN** *Themeda* **AND** *Digitaria*

Both species documented in this study were within the typical silica range for dryland grasses, but *Themeda* had significantly greater silica content than *Digitaria.* These two species have different suites of anatomical and physiological traits and may be considered distinct graminoid functional types (Coughenour, 1985; Diaz and Cabido, 1997). Lawn grasses, such as *Digitaria*, exhibit clonal, stoloniferous growth; their prostrate growth form allows them to reduce tissue loss to herbivores while quickly spreading horizontally to form lawns under high soil fertility (Cromsigt and Olff, 2008). In contrast, the higher lignin content of tall, dense, and slow-growing bunch grass species, like *Themeda*, makes them less palatable to herbivores than lawn grasses (Sinclair and Norton-Griffiths, 1979). Thus, as a consequence of their different life history strategies, it is not surprising that these two species differ in the degree to which silica accumulates in their leaves.

As a documented growth promoter (McNaughton et al., 1985; Isa et al., 2010) and metabolically "cheap" structural substitute for carbon-based compounds such as lignin (Raven, 1983; Cooke and Leishman, 2011a), silica deposition may provide an alternative mechanism for accelerated growth which would prove especially beneficial for bunch grasses. The increased growth rates that result from silica accumulation may substantially improve light interception of slow-growing species which primarily compete for sunlight (Ando et al., 2002). This idea is further supported by a positive correlation between leaf length and silica content observed in *Spartina* (Querné et al., 2012), again supporting the idea that silica allows improved growth without requiring significant carbon investment (Cooke and Leishman, 2012). Further, slow-growing bunch grasses display greater leaf mass per area, LMA, (Fynn et al., 2011) and leaf dry matter content, LDMC, (Anderson et al., 2013) than fast-growing lawn species, indicating a greater relative investment in dry matter. The worldwide leaf economic spectrum (Wright et al., 2004) predicts a strong positive correlation between dry matter investment and leaf longevity, suggesting that, based on their higher LMA, bunchgrass species likely also have leaves of greater longevity than lawn-grass species. Thus, it may be beneficial for relatively longlived bunch grasses to invest in immobile phytoliths (Endara and Coley, 2011) which allow for rapid augmentation of cell structure of C-limited species under intense light competition. On the other hand, recent research revealed a negative correlation between leaf lifespan and Si concentration when considering a broad range of plant functional types (Cooke and Leishman, 2011b). The reasons for differential responses to defoliation and high interspecific variation in this qualitatively ubiquitous defense within Poaceae remain unclear. Here we focused on two species representing extremes of each of the two grass growth forms (bunch, lawn); greater sampling from along the gradient of short to tall grasses is needed to better understand the influence of these contrasting physiological forms on silica accumulation.

#### **DEFOLIATION RESPONSE**

Contrary to our initial predictions, clipping did not result in silica induction for either the lawn or bunch grass species, with nearly identical mean silica content observed for defoliated and non-defoliated individuals. There are several possible explanations for the lack of silica induction observed in our Serengeti common garden study. First, the frequency and/or intensity of defoliation may not have been great enough to elicit a response. For example, in the Serengeti plains, more than 1 million migratory ungulates are present in dense herds during the wet season, and localized grazing "hotspots" support dense spatially and temporally stable grazer communities that inflict especially frequent and intense herbivory on plant communities (Holdo et al., 2009; Anderson et al., 2010). Our literature review further corroborates this notion. For example, a laboratory study by Massey et al. (2007) revealed that a single defoliation event did not induce silica uptake, while repeated defoliation by herbivores (16 events total) significantly increased silica content of ryegrass and fescue, revealing the importance of both frequency and duration of defoliation. Extent of tissue removal (5% vs. 25%) is also proven to affect both induction and relaxation of silica response, demonstrating a threshold effect required for induction (Reynolds et al., 2012). In our study, grasses were defoliated on a bi-monthly basis, four times total. For at least certain areas of Serengeti, this may be lower than levels of natural grazing intensity due to Serengeti's rich herbivore fauna.

In addition, manual defoliation (i.e., clipping) likely does not elicit the same response as natural grazing under laboratory or field conditions, a well-known effect with respect to induced defenses. Our literature review revealed that clipping and grazing both resulted in silica induction in ∼50% of the studies (5 of 11 clipping cases, 12 of 23 grazing cases), but the magnitude of this response was much greater under grazing defoliation, as illustrated in **Figure 4**. Among grazing studies, silica induction was observed in response to insect, small mammal, and large mammal grazing. We propose that clipping may not be sufficient to represent both the direct and indirect effects of grazing on silica dynamics in grasses; this may be especially true for grass species growing in the Serengeti plains, which have a long co-evolutionary history with herbivores. Large-bodied grazers compact soils, resulting in increased bulk density (e.g., Bakker et al., 2004; Holdo and Mack, 2014; Veldhuis et al., 2014) and matric water potential and, thus, alter the ability of roots to absorb water. While manual defoliation studies yield information about certain physiological responses, the literature review presented here suggests that this approach may not properly mimic the complex physiological responses associated with natural grazing, some of which may result from indirect effects associated with modification of soil water (see below). In summary, plants perceive herbivore effects through a suite of signals, some of which, such as changes in the abiotic environment, are not elicited directly by the removal of tissues.

#### **WATER AND COLLECTION SITE EFFECTS**

*Themeda* individuals in the high water treatment accumulated significantly greater foliar silica than those in the ambient water group (**Figure 2**), but this trend was not observed for *Digitaria*. This suggests either differential silica uptake mechanisms of the two species or different generalized physiological responses to drought which indirectly influence silica accumulation. Since *Themeda* responded to changes in water availability, we hypothesize that active uptake mechanisms are present in this species which allow it to increase uptake of Si(OH)4 from the soil solution when water is abundant. If true, this then raises the question, why would *Digitaria* not also increase uptake of a ubiquitously beneficial element under similar conditions? One possible explanation is that the different life histories of these two species, coupled with the proposed costs of silica accumulation (Cooke and Leishman, 2012) have resulted in selection for differential silica uptake mechanisms, in which tall, bunch species but not short, lawn species, have incorporated active Si uptake mechanisms such as the use of specialized energy-dependent transport molecules. *Digitaria* has highly flexible leaves which are capable of curling under drought conditions to prevent transpiration; this drought tolerance mechanism would likely be hindered due to the loss of bulliform cell function associated with leaf tissue silicification (Honaine and Osterrieth, 2011). Conversely, the benefit of improved drought tolerance associated with silica accumulation (Gao et al., 2006; Eneji et al., 2008) is likely of greater relative importance for bunch grass species, such as *Themeda*, which experience intense light competition and a significant seasonal water deficit. Increased silica uptake under high water availability may serve to buffer bunch grasses against future drought events while simultaneously producing an erect canopy structure (see Isa et al., 2010) for light competition and offsetting carbon costs. Under this proposed hypothesis, that bunch grasses benefit more than lawn grasses due to more intense light competition, any anti-herbivore benefits are secondary to the direct adaptive significance of silica accumulation, which is related to water and/or light limitation

Consistent with the observed effects of watering treatments, the silica differences among collection sites also indicate that

water availability may be an important determinant of grass foliar silica content. *Themeda* individuals collected from high rainfall sites KCW and MSB, where light competition is expected to be more intense, exhibited significantly greater silica content than individuals collected from the low rainfall site TOG, where light competition is expected to be less intense. In contrast, the leaf silica content of *Digitaria* individuals was not influenced by collection site (**Figure 3**). This observation reinforces the notion that water strongly influences foliar silica content of grasses and implies a pattern of increasing silica content of tall bunch grass species, like *Themeda*, with increasing distance from the plains and increasing light competition.

Other possible explanations exist for the observed site-level variation in silica content of *T. triandra*. Numerous fertilization studies have indicated that plant silica content is strongly correlated with availability of Si(OH)4 in the soil medium (Fox et al., 1969; Jones and Handreck, 1969; Van der Vorm, 1980; Gali and Smith, 1992), and more recently, ecological studies have demonstrated a similar relationship between soils and plants in natural systems. Biogenic silica content of grasses in a South African savanna was higher for plants collected from basaltic soils than for those collected from granitic soils, reflecting differences in weatherability and dissolved silica (DSi) content of these contrasting parent materials (Melzer et al., 2011). Cooke and Leishman (2012) compared foliar silica content of plant communities from Hawkesbury sandstone and a nearby diatreme forest with nearly three times as much plant available soil Si; though not statistically significant, they observed a general trend of higher foliar silica content for plants from the diatreme site, including a fivefold difference in leaf silica observed for the sedge *Schoenus melanostachys*. The unique nature of the silica-depleted carbonatite ash that enriches the Serengeti plains may contribute to a gradient in available soil Si, in which available Si increases toward the northern and western corridors of the park. However, more work is needed to document how soil Si availability varies within SNP and how it may interact with gradients in soil moisture, pH, and other important soil nutrients known to vary across the landscape (de Wit, 1978; Jager, 1982). Finally, we caution that, due to geographic separation, plant genotype should also vary among sites (Soininen et al., 2013), and we cannot rule out genotypic variation as an important influence on silica accumulation patterns.

#### **ECOLOGICAL AND EVOLUTIONARY IMPLICATIONS**

The results of our literature review suggest that induced silica response to defoliation is dynamic and dependent upon interactions with additional biotic and abiotic factors. For example, defoliation by burning resulted in more than a 1/3 decrease in silica content of *Aristida* grass (Dufek et al., 2014). Moreover, seasonal (Georgiadis and McNaughton, 1988, 1990; Carey and Fulweiler, 2013; Struyf et al., 2005) and inter-annual (Morton and Jutras, 1974; Pahkala and Pihala, 2000) variations in field measurements of grass silica content indicate that leaf tissue silicification is highly plastic. While seasonal fluctuations in grass silica content are welldocumented, it remains unclear whether dynamic silica values measured in the field occur in response to changes in grazing intensity, temperature, precipitation, or, most likely, a complex interaction among these stochastic variables.

Silica accumulation is considered a central axis of grass–grazer coevolution throughout the literature, often cited as a driving force behind the evolution of hypsodonty and the coupled taxonomic radiation of grasses and grazers (Mendoza and Palmqvist, 2008; Hummel et al., 2011). The evolution of large-bodied mammalian grazers is thought to have selected for specialized traits of the Poaceae such as meristematic growth from a well-protected crown, rapid growth rate, and high levels of Si accumulation (McNaughton, 1979; Coughenour, 1985). Likewise, these plant traits are thought to have influenced herbivore dentition, digestion, and behavior (McNaughton, 1984; Massey and Hartley, 2006; Erickson et al., 2012). Our study and literature review, however, suggest that the evolutionary significance of silica accumulation may be a remnant of tradeoffs associated with the leaf economic spectrum that resulted in the evolution of two contrasting grass functional types. Silica accumulation may have first served to improve drought tolerance with herbivore deterrence as a secondary role. Fossil data support this notion: grassdominated habitats preceded the appearance of North American and Mediterranean grazing specialists (Strömberg, 2006; Strömberg et al., 2007), and an investigation of the Gran Barranca fossil record indicates that open grass habitats were not necessary pre-conditions for favoring early hypsodont mammals in Earth's earliest grass-dominated systems (Strömberg et al., 2013).

### **ACKNOWLEDGMENTS**

The authors thank Dr. George Donati of WFU for assistance with ICP measurements of silica. We also thank the Serengeti Wildlife Research Centre, Tanzania Wildlife Research Institute, Tanzania National Parks, and the management of Serengeti National Park for their help during our experiments. We offer a special thanks to Emilian P. Mayemba for helping with the construction of the plot exclosures and to Peter M. Ezra for assistance with the common garden study. Financial support for this project was supported in part by an Early Career Project Grant from the British Ecological Society (UK) to T. M. Anderson and grant DEB-1145861 from the National Science Foundation (USA) to T. M. Anderson.

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fpls.2014.00568/ abstract

**Table S1 | Results from linear mixed model in R testing for interactions between species and water on foliar silica accumulation.** Block was included as a random effect. P values in bold indicate significance at α = 0.05.

**Table S2 | Summary of studies included in literature review of induced silica response of Poaceae; colors in the "Defoliation Response" column correspond with Figure 4.** In the Defoliation Response column, "0" indicates that a species exhibited less than a 20% relative change in foliar silica content in response to defoliation, while "+" indicates a ≥ 20% increase in silica, and a "−" indicates a ≥ 20% decrease in silica.

#### **REFERENCES**

Honaine,M. F., and Osterrieth,M. L. (2011). Silicification of the adaxial epidermis of leaves of a panicoid grass in relation to leaf position and section and environmental conditions. *Plant Biol.* 14, 596–604. doi: 10.1111/j.1438-8677.2011.00530.x

Hothorn, T., Bretz, F., and Westfall, P. (2008). Simultaneous inference in general parametric models. *Biom. J.* 50, 346–363. doi: 10.1002/bimj.200810425

Hummel, J., Findeisen, E., Südekum, K., Ruf, I., Kaiser, T. M., Bucher, M., et al. (2011). Another one bites the dust: faecal silica levels in large herbivores correlate with high-crowned teeth. *Proc. R. Soc. B Biol. Sci.* 278, 1742–1747. doi: 10.1098/rspb.2010.1939

Sangster, A. G., and Parry, D. W. (1970). Silica deposition in the grass leaf in relation to transpiration and the effect of dinitrophenol. *Ann. Bot.* 35, 667–677.

White, F. (1983). *The Vegetation of Africa.* Paris: UNESCO.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 04 June 2014; accepted: 02 October 2014; published online: 21 October 2014. Citation: Quigley KM and Anderson TM (2014) Leaf silica concentration in Serengeti grasses increases with watering but not clipping: insights from a common garden study and literature review. Front. Plant Sci. 5:568. doi: 10.3389/fpls.2014.00568*

*This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Quigley and Anderson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Defending the leaf surface: intra- and inter-specific differences in silicon deposition in grasses in response to damage and silicon supply

# *Sue E. Hartley\*, Rob N. Fitt, Emma L. McLarnon and Ruth N. Wade*

Department of Biology, University of York, York, UK

#### *Edited by:*

Julia Cooke, University of Western Sydney, Australia

#### *Reviewed by:*

Francesco De Bello, Czech Academy of Sciences, Czech Republic Caroline A. E. Stromberg, University of Washington, USA Anthony Joern, Kansas State University, USA

#### *\*Correspondence:*

Sue E. Hartley, Department of Biology, University of York, Wentworth Way, Heslington, York YO10 5DD, UK e-mail<EMAIL_ADDRESS>
Understanding interactions between grasses and their herbivores is central to the conservation of species-rich grasslands and the protection of our most important crops against pests. Grasses employ a range of defenses against their natural enemies; siliconbased defenses have been shown to be one of the most effective. Silicon (Si) is laid down on the leaf surface as spines and other sharp bodies, known as phytoliths, making grasses abrasive and their foliage indigestible to herbivores. Previous studies on Si defenses found that closely related species may have similar levels of Si in the leaves but differ markedly in abrasiveness. Here we show how the number, shape and distribution of Si-rich phytoliths and spines differ within and between different grass species and demonstrate that species also differ in their ability to change the deposition and distribution of these defenses in response to damage or increases in Si supply. Specifically, we tested the response of two genotypes of Festuca arundinacea known to differ in their surface texture and three different grass species (F. ovina, F. rubra, and Deschampsia cespitosa) differing in their abrasiveness to combined manipulation of leaf damage and Si supply. F. arundinacea plants with a harsh leaf surface had higher Si content and more spines on their leaf surface than soft varieties. F. ovina and D. cespitosa plants increased their leaf Si concentration and produced an increase in the number of leaf spines and phytoliths on the leaf surface in response to Si addition. F rubra also increased leaf Si content in response to treatments, particularly in damaged leaves, but did not deposit this in the form of spines or increased densities of phytoliths. We discuss how the form in which grasses deposit Si may affect their anti-herbivore characteristics and consider the ecological and agricultural implications of the differences in allocation to Si-based defenses between grass species.

**Keywords: plant defense, silicon, abrasion, grasses, leaf surface, phytoliths, spines**

#### **INTRODUCTION**

Grasslands including managed rangelands and pastures cover ∼40% of the earth's surface and grasses are an important plant family agriculturally, economically and ecologically (Strömberg, 2005; Gibson, 2009). Not only are our most widely grown and consumed food crops domesticated grass species, but grasses also provide grazing for both wild and domesticated animals. In their long co-evolution with grazers (Coughenour et al., 1985), grasses have developed a number of defensive strategies to both tolerate and repel herbivory (Vicari and Bazely, 1993), including rapid regrowth ability from their basal meristems (also an adaptation to fire and trampling common in these ecosystems) and a combination of both chemical defenses (including those provided by endophyte mutualists; Hartley and Gange, 2009) and physical defenses (McNaughton and Tarrants, 1983).

One such physical defense is the accumulation of silicon (Si) which has been previously reported to accumulate in high levels in the leaves of many grass species (Hodson et al., 2005), although the amount of Si accumulated shows large inter and intra species variation (Massey et al., 2007a; Soininen et al., 2013). There is clear evidence to demonstrate that these high levels of Si are effective anti-herbivore defenses, with impacts on the feeding preferences and performance of both vertebrate (McNaughton and Tarrants, 1983; Gali-Muhtasib et al., 1992; Massey and Hartley, 2006; Teaford et al., 2006; Massey et al., 2009) and invertebrate herbivores (Goussain et al., 2005; Massey et al., 2006; Kvedaras et al., 2007; Massey and Hartley, 2009).

These adverse effects appear to be mediated at least in part by abrasion: Si is primarily deposited as amorphous silica in the form of solid bodies known phytoliths in the epidermis (Richmond and Sussman, 2003; Currie and Perry, 2007). Phytoliths are hard and often irregular shapes and Si is also deposited in leaf hairs, trichomes and spines; all these structures could influence the texture and abrasiveness of the leaf. It has been suggested that Si abrades the teeth of mammalian herbivores (Jernvall and Fortelius, 2002; Erickson, 2013 but see Sanson et al., 2007) and an increase in leaf abrasiveness has been shown to reduce the performance of both vertebrate and invertebrate herbivores. For example, the amount of mandible wear feeding imposed on African armyworm (*Spodoptera exempta* Walker), and hence the reduction in their ability to extract nitrogen from their food, is correlated with the Si levels of the foliage they consume (Massey and Hartley, 2009), whilst voles prefer, and perform better on, grasses which are less abrasive (Massey and Hartley, 2006; Massey et al., 2008).

Previous work has suggested that foliar Si levels and the abrasiveness of grass leaves are reasonably well correlated: over 70% of the variation in abrasiveness across 18 different grass species was explained by Si content (Massey et al., 2007a). However, Si levels and abrasion are not always closely linked. For example, despite containing similar concentrations of Si, *Festuca ovina* L. was found to have much higher levels of abrasiveness compared to *F. rubra* L., whilst increasing leaf Si concentration through Si addition produced a smaller increase in abrasiveness in *Poa annua* L., a relatively palatable species, than in the more unpalatable *Brachypodium pinnatum* (L.) P. Beauv. (Massey et al., 2007a). It is possible that different grass species deposit their available Si differently at their leaf surfaces, influencing the abrasiveness of their leaves. It is certainly well-known that phytolith morphology varies between plant taxa, with differences between species sufficiently marked and consistent to allow phytoliths to be useful in palaeobotany (Strömberg, 2005). Some phytoliths are relatively smooth in shape, others much less so and it seems likely that the size, shape and density of phytoliths and Si rich spines will influence the abrasiveness of the leaf surface and its impact on the preferences and performance of herbivores.

Another influence on the nature and effectiveness of the leaf surface defenses will be the amount of Si available in the soil to take up and deposit (Currie and Perry, 2007). Previous exposure to herbivory has also been shown to impact on the levels of Sibased defenses in plants. It has long been known that Si levels increase in grasses from grazed areas (McNaughton and Tarrants, 1983) and herbivore-specific induction in Si defenses has been shown to occur, but only after repeated damage above a threshold (Massey et al., 2007b; Reynolds et al., 2012). More recently it has been shown that there are differences in both grass species and grass genotypes in the extent to which they respond to damage with increased Si uptake (Soininen et al., 2013).

The aim of this study was to determine the leaf Si concentration of different forage grass genotypes and naturally occurring grass species previously reported to differ in their leaf abrasiveness (see below), and to investigate whether these differences in leaf texture are related to the way Si is deposited on the leaf surface, potentially influencing the effectiveness of their use of Si in terms of reducing palatability to herbivores. We hypothesized that:

(i) harsher and more abrasive species and varieties would have higher leaf Si levels than softer ones;

(ii) species with similar Si levels which differed in abrasiveness would do so because they used their Si to produce a greater number of phytoliths and/or spines on their leaf surface, and that these spines would be larger or sharper.

We also hypothesized that irrespective of grass species, foliar Si levels would be elevated by increases in Si supply, and hence in potential uptake (Epstein, 1999; Cooke and Leishman, 2011), and by damage, due to induction (Massey et al., 2007b). We also expected foliar Si levels to be highest in plants receiving both Si addition and damage, since induction in response to damage would be able to capitalize on the additional Si available in the soil. We predicted that the most abrasive species would deposit this additional Si in the form of surface spines to a greater extent than less abrasive species.

# **MATERIALS AND METHODS**

# **STUDY SPECIES**

*Festuca arundinacea* Schreb. is a cool season perennial grass (Gibson and Newman, 2001) and a dominant pasture and turf grass in North America, Australia and Europe (Hand et al., 2012). It has a number of attractive agronomic attributes, including high yields, winter persistence (Gibson and Newman, 2001) and tolerance to drought (Cougnon et al., 2014), though it appears to be relatively unpalatable to cattle. In mixed culture fields, cattle rarely choose it as their forage choice (Gibson and Newman, 2001), possibly because of the"harsh" (i.e., feeling rough to the touch) texture of the leaf surfaces. There is interest amongst forage breeders in understanding the basis of this leaf harshness and unpalatability to improve the attractiveness of this species as forage. A number of varieties of *F. arundinacea* ranging from very harsh to very soft leaf textures have been developed by plant breeders (based on manual evaluation of surface roughness in the field by plant breeders), enabling the testing of the hypothesis that Si has a role in causing the harsh leaf surfaces.

We can also address the relationship between Si content and leaf texture by exploiting the natural variation in the relationship between Si and abrasion across native non-forage *Festuca* species: *F. ovina* and *F. rubra* may differ so markedly in their leaf abrasion despite similar foliar Si levels (Massey et al., 2007a) because of the way they utilize the Si they take up. Specifically, *F. ovina* may produce a greater number, larger or more abrasive spines and phytoliths than *F. rubra*. We compared these species with the Si content and leaf texture of *Deschampsia cespitosa*, a grass known to be particularly unpalatable to herbivores due to its Si defenses (Massey and Hartley, 2006, 2009).

#### **PLANT GROWTH AND EXPERIMENTAL TREATMENTS**

The two varieties of *Fesctuca arundinacea* were grown individually from seed in a loam based compost (John Innes No.2) in 13 cm pots. Both varieties were harvested 8 weeks after sowing at the point where all plants had at least four tillers. *F. ovina, F. rubra*, and *D. cespitosa* (L.) were grown from seed individually in peat based F2 (Levington, Scotts) compost in 10 cm diameter pots in the greenhouse conditions 16 h daylight, 20◦C/15◦C day/night. Due to their slower growth rates in relation to *F. arundinacea*, these three species were harvested 18 weeks after sowing.

Once established, plants were randomly assigned to four treatments: control plants with no Si addition and no damage (Si-D-), Si addition only (Si+ D-), damage only (Si- D+), and both Si addition and damage (Si+ D+). Treatments were imposed 3 weeks after sowing in the case of *F. arundinacea*, with plants harvested 5 weeks later, and 8 weeks after sowing in the case of the other three species, which were harvested 10 weeks later. There were six replicate plants of each treatment combination for *F. arundinacea* and seven replicate plants of each treatment for the other species.

For all grass species and varieties, Si addition was achieved by watering plants with 150 mg L−1 solution of dissolved sodium metasilicate (Na2SiO3·9*H*2O). Plants were watered 100 ml twice a week with either Si solution or deionised water. *F. ovina, F. rubra*, and *D. cespitosa* plants in the two treatments where damage was applied were mechanically damaged using scissors once a week over 10 weeks. Half of the plant's leaves were damaged by removing approximately half the leaf lamina down the midrib; the remaining leaves were left undamaged. When damaged plants were harvested, damaged and undamaged leaves were kept separated for Si analysis in order to test for induction of Si defenses in both the undamaged and the damaged leaves on the damaged plants.

#### **Si ANALYSIS BY PORTABLE X-RAY FLUORESCENCE SPECTROMETRY (P-XRF)**

Silicon was analyzed by P-XRF, calibrated using Si-spiked synthetic methyl cellulose and validated using Certified Reference Materials of NCS ZC73014 'Tea' obtained from China National Analysis Center for Iron and Steel (Reidinger et al., 2012).

Both P-XRF and EDX (see below) work on the principle of excitation of inner orbital electrons by an X-ray radiation source. As the excited electrons relax to the ground state, they fluoresce, thereby ejecting photons of energy and wavelength characteristic of the elements present and their concentrations. XRF instruments are widely used for the non-destructive, rapid and accurate elemental analysis of a range of materials (Jang, 2010).

Leaf material was ball milled (Retsch MM 400, Haan, Germany) for 2 min at a vibrational frequency of 24 Hz (60 min−1) with two 1 cm diameter steel grinding balls in a 25 ml grinding jar. Leaf material was pressed at 11 tons for approximately 5 s into 5 mm thick cylindrical pellets with a manual hydraulic press using a 13 mm die (Specac, Orpington, UK). Si analysis (% Si DW) was performed using a commercial P-XRF instrument (Nitron XL3t900 GOLDD analyser: Thermo Scientific Winchester, UK) held in a test stand (SmartStand, Thermo Scientific, Winchester, UK).

#### **SURFACE ANALYSIS BY SCANNING ELECTRON MICROSCOPY (SEM) AND ENERGY DISPERSIVE X-RAY SPECTROSCOPY (EDX)**

Leaf samples were taken from two replicate plants per species from all four treatment combinations for the inter-species experiment. A square section (∼5 mm2) of leaf material either side of the midrib of a mature, expanded leaf blade on the main stem was cut with a razor blade and immediately placed in fixative (2.5% glutaraldehyde, 4% formaldehyde in 100 mM phosphate buffer). For the *F. arundinacea* experiment, samples were taken from the harsh variety with added Si and the soft variety with no added Si (Si addition had no effect on Si levels in this experiment – see below). A square section (∼1 cm2) spanning the entire width of the mature, expanded leaf blade was cut from the main stem for each variety. The samples were then dehydrated through an acetone graduated series (samples were placed at 25, 50, 75, and 100% acetone concentration for ∼1 h) and critical-point dried. Samples were then mounted on sticky carbon tabs and coated with 8nm thick layer of platinum-palladium.

SEM images were obtained using FEI Sirion S-FEG FESEM (Oxford Instruments, Tubney Woods, Abingdon, Oxfordshire). EDX was used in conjunction with the SEM to determine the elemental composition of the samples; an electron beam was focused on the samples and the difference between the ground state (unexcited state) and the excited state was measured by the energydispersive spectrometer which determines the elements present in the sample (Goldstein, 2003). The EDX analysis was performed using an Oxford INCA analysis system FESEM (Oxford Instruments, TubneyWoods,Abingdon, Oxfordshire), using the working distance of 10 mm. For the SEM images, the voltage was 5–10 kV and for the EDX analysis the voltage was 12 kV.

#### **STATISTICAL ANALYSES**

All analyses were performed using R (version 3.0.2). ANOVA was used to test the main and interactive effects of grass species or genotype, Si addition and damage treatments on leaf Si concentrations. The effects of the Si and damage treatments were assessed on undamaged leaves from plants across all four treatments, to test if damage led to increased Si levels systemically in damaged plants in comparison with undamaged plants. A separate analysis tested for the effect of these treatments in damaged leaves from damaged plants compared to undamaged leaves from undamaged plants. *Post hoc* Tukey contrast tests were performed using the ghlt function from multcomp package (Hothorn et al., 2014).

Linear models were used to check for normality and homogeneity of variance following Crawley (2007). Si (%) values were transformed using the arcsine squareroot transformation to meet the assumptions of the test. Significance was set at *P* < 0.05 for all analyses.

# **RESULTS**

#### **INTRASPECIFIC DIFFERENCES**

The *F. arundinacea* variety with a harsh leaf surface texture had significantly higher leaf Si concentration than the soft texture variety (*F*1,19 = 8.586, *P* < 0.01), but there was no significant interaction between Si addition and variety (*F*1,19 = 0.282, *P* > 0.5; **Figure 1**).

and soft variety abaxail margin (x150 magnification). Red circles indicate leaf spine presence.

The two main types of cells which were silicified were leaf spines (trichomes) and silica short cells. The harsh variety had more spines present on the abaxial surface than the soft variety (**Figures 2A,B**), which not only had fewer spines but the spines which were present were smaller and had a different morphology (**Figures 2B,C**). The spines present on the harsh variety were bigger in size and the point of the spines were spear-like in appearance; these spines also appeared to protrude more from the surface compared with the soft variety, where the spines were smaller in size and the points of the spines lay closer to the surface of the leaf. No spines were observed on the adaxial surface of either variety of *F. arundinacea* (images not shown).

The spines were rich in Si (**Figure 3**), and there were other Si deposits on the leaf surface in the form of silica short cells. Generally, the harsh variety had a greater over surface deposition of Si compared with the soft one, depositing the Si within the leaf spines (red circles **Figure 3**), and also silica short cells surrounding the spines (red circles **Figure 3**). The soft variety deposited Si as silica short cells on the leaf surface within fewer, smaller leaf spines containing less Si than in the harsh variety (**Figure 3B**).

#### **INTERSPECIFIC DIFFERENCES**

Grass species differed in their leaf Si concentration (**Figure 4**; Spp effect: *F*2,72 = 23.62, *P* < 0.001 undamaged leaves; *F*2,72 = 15.99,

*P* < 0.001 damaged leaves) with significantly lower Si concentrations in *D. cespitosa* compared to *F. rubra (post hoc* Tukey tests *P* < 0.05 for both undamaged and damaged leaves) and *F. ovina (post hoc* Tukey tests *P* < 0.01 for both undamaged and damaged leaves*).* Plants treated with Si addition responded with an increase in their leaf Si concentration irrespective of whether leaves were damaged or not (Si effect *F*1,72 = 1265.33, *P* < 0.001 undamaged leaves; *F*1,72 = 463.17, *P* < 0.001 damaged leaves). In

comparison with undamaged leaves on undamaged plants, damage did not increase Si levels in undamaged leaves on damaged plants (*F*1,72 = 0.03, *P* > 0.05 NS), but there was a significant increase in the Si levels in the damaged leaves (*F*1,72 = 17.92, *P* < 0.001), suggesting that damage-induced increases in Si levels are localized in damaged leaves and do not spread to undamaged ones on the same plant (**Figure 4**).

In the case of undamaged leaves there was no significant interaction between the effects of species and Si addition on Si levels (*F*2,72 = 2.89, *P* > 0.05 NS), suggesting all three species responded in a similar way to increases in Si supply in terms of the allocation of this additional Si to their undamaged leaves. However this was not the case for damaged leaves, where a significant Species × Si addition interaction (*F*1,72 = 4.62, *P* < 0.05) suggests species differ in where they allocate any additional Si once they are damaged. This is confirmed by the *post hoc* Tukey tests which showed that *F. rubra* had significantly higher concentrations of Si in damaged leaves under conditions of increased Si supply than either *F. ovina* (*P* < 0.01) or *D. cespitosa* (*P* < 0.001; **Figure 4**).

The SEM revealed differences in Si deposition on the leaf surfaces of the grass species (**Figure 5**). The leaf surface of *D. cespitosa* plants was found to have abundant Si-rich leaf spines (trichomes), even in the absence of Si addition. In contrast, the leaf surface of *F. rubra* and *F. ovina* plants growing without added Si had only rounded silica short cells and no leaf spines, although the round phytoliths were much more prominent and frequently distributed on the leaf surface of *F. ovina* than *F. rubra*. Both *F. ovina* and *D.*

*cespitosa* plants deposited additional phytoliths (silica short cells) in response to increased Si supply, especially in the presence of damage, but Si addition had very little effect on the number or shape of the phytoliths deposited on the leaf surface of *F. rubra*. The damage alone treatments had little effect on leaf surface Si deposition in any of the grass species (images not shown).

# **DISCUSSION**

Grass species and varieties differed in their leaf Si concentrations and the form in which this Si is deposited on the leaf surface. The more unpalatable and abrasive species, namely *D. cespitosa* and the harsh variety of *F. arundinacea* had both larger and a greater abundance of Si-rich spines compared to the more palatable *F. rubra* and soft variety of *F. arundinacea*. Si addition resulted in an increase in leaf Si concentration in three out of the four grass species and altered the deposition of Si on the leaf surface in the case of *D. cespitosa* and *F. ovina*, but had little impact on the surface of *F. rubra*. The different forms in which Si is deposited at the leaf surface in *F. ovina* and *F. rubra* may explain previous observations that they differ in abrasiveness even though, as we found here, they contain similar amounts of Si. Generally, damage caused a small increase in leaf Si concentration, but did not have a large effect on the form in which the Si was deposited on the leaf surface.

# **INTRASPECIFIC DIFFERENCES**

As we hypothesized, the harsh variety of *F. arundinacea* had a higher leaf Si level than the soft variety, though our prediction

that Si addition would increase foliar Si content was not supported in this species. The differences in foliar Si content between the harsh and soft varieties suggest that Si is contributing significantly toward the differences in leaf texture between them. The harsh variety had significantly higher leaf Si content than the soft variety as well as having a different leaf surface morphology, suggesting that increased levels of Si support the production of increased number and size of leaf spines. This is significant in the context of forage grass: differences in the patterns of deposition of Si on the leaf surface between varieties may offer scope for plant breeders to select for more palatable forage.

Scanning electron microscopy images also revealed that the harsh variety of *F. arundinacea* had more numerous and larger leaf spines than those on the leaf surface of the soft variety, and this was particularly noticeable at the leaf margin. These spines may act as a deterrent to herbivores, especially cattle as they use their tongues to wrap around the blades of grass prior to chewing; if the grass feels spiny then it is likely to seem unpalatable to the cattle. An impact of Si levels on the bite rate of ruminants has been demonstrated experimentally for sheep (Massey et al., 2009), suggesting Si does impair food processing time. The soft variety had far fewer and smaller spines, suggesting these plants are depositing Si in a different way to the harsh variety. Even when not deposited as abrasive spines, Si can still make plants hard to digest, if for example these deposits prevent herbivores crushing cells to extract nutrients, as hypothesized by Hunt et al. (2008).

Contrary to our predictions, neither variety responded to the Si addition treatment with an increase in leaf Si content. This may be related to the young age of the plants and relatively short duration of the Si addition treatment. Si accumulation is influenced by transpiration rates, where older leaves are found to have significantly more Si than younger leaves, primarily due to Si translocation via the transpiration stream (Piperno, 2006). Furthermore, once deposited Si is not remobilized (Richmond and Sussman, 2003), meaning foliar Si levels increase with both plant and leaf age (Reynolds et al., 2012). Although the mechanisms underpinning Si uptake and distribution in plants are still not fully understood, it has been demonstrated that plant species differ in the Si uptake ability of their roots and in the density of their root transporters, as well as in their capacity to upload Si to the xylem (Ma and Yamaji, 2006). More recently, work on rice has suggested that shoots control the regulation of the Si transporters in the root and hence how much Si is taken up into the shoot (Yamaji and Ma, 2011). A study assessing the uptake abilities of over 500 plant species (Ma and Takahashi, 2002) classified them into high, intermediate and nonaccumulators and it may be that *F. arundinacea* physiology is such that it is not a high accumulator of Si, even under conditions of high Si supply. It does however, appear to be able to use the Si it does take up very efficiently in terms of spine production, at least in the case of the harsh variety. The mean Si values reported here for *F. arundinacea* are lower than those reported for this species in Hodson et al. (2005), which may reflect differences in age of the plants when sampled or the growing conditions of the plants. However the variation in foliar Si content shown in this species and indeed in other taxa within the genus (Hodson et al., 2005; Massey and Hartley, 2006; Massey et al., 2006, 2007a, 2009) suggests a high degree of phenotypic plasticity in the levels of Si seen

within the leaves of *Festuca* species. This is perhaps unsurprising given the numerous factors, including plant genotype, biotic stresses such as herbivory and abiotic ones such as water availability, known to affect Si levels in plants (Soininen et al., 2013; Quigley and Anderson, 2014).

### **INTERSPECIFIC DIFFERENCES**

Our hypothesis that abrasive species would have higher foliar Si concentration than less abrasive species was not well-supported: *D. cespitosa* has previously been reported to have high leaf Si and to be more abrasive than either of the *Festuca* species, but in this study had lower leaf Si concentration than *F. ovina* and *F. rubra*. Differences in experimental conditions, and hence in plant growth rate, and in plant age, size, and genotype (Soininen et al., 2013) may explain changes in the relative Si concentrations between species, but it is clear that our second hypothesis, namely that more abrasive species had larger, sharper and/or a greater number of spines and phytoliths is supported (**Figure 5**). SEM images revealed that *D. cespitosa* leaves are covered in Si-rich leaf spines which were absent from the leaf surfaces of *F. rubra* (and from *F. ovina* in the absence of additional Si). This strongly suggests that the leaf spines were significantly influencing the abrasiveness of *D. cespitosa* and that phytolith morphology may be more important than leaf Si concentration in determining the abrasiveness of leaves and thus the effectiveness of anti-herbivore defense.

There was a change in morphology and an increase in the number of Si-rich bodies deposited on the leaf surface of *D. cespitosa* and *F. ovina* when plants were provided additional Si; in the case of *D. cespitosa*, phytoliths which had not been present in control leaves were deposited (silica short cells), whereas for *F. ovina*, new, Si-enriched spines were produced, again when spines were not apparent on control leaves. This suggests that these plant species have the ability to deposit new types of Si-based structures to potentially increase their anti-herbivore defenses, whether via abrasion, digestibility effects or both, when Si supply is increased. These changes were most obvious when leaves were also damaged, although interestingly damage in the absence of additional Si did not produce them. In addition to changes in the nature of the spines, the EDX demonstrates that *D. cespitosa* deposited Si only at the tips of spines under control conditions, but under the Si addition treatment, the spines contain Si throughout and the leaf surface is also heavily silicified. A similar pattern was observed in *F. ovina* (**Figure 5**).

Our results support our predictions about the influence of Si supply on the level of Si-based defenses (also see Garbuzov et al., 2011), but damage had less effect on Si-based defenses than we predicted. Although damaged plants were found to have an increase in leaf Si concentration, this was far smaller than the effect of Si supply and there was little effect of damage on spine formation. This may reflect the fact that we used a clipping treatment; simulated damaged may not bring about the same response in spine/phytolith morphology and Si accumulation as herbivory, as reported in previous studies (Massey et al., 2007b). *F. rubra* demonstrated a greater Si uptake to damaged leaves on damaged plants than other species did, which suggests plant species show differences in the way they distribute their Si between different plant parts in response to damage (and potentially other stresses).

The mechanism for this is currently unknown, but there have been reports of between species differences in the ability to load Si into the xylem (Ma and Yamaji, 2006).

# **CONCLUSION**

There were marked differences in the way that even grass species from the same genus deployed the Si they take up in terms of its deposition in structures likely to affect their anti-herbivore defenses. Differences in the localization and the Si-based structures formed has been demonstrated before between plant families (Currie and Perry, 2007), but to our knowledge this is the first time such striking variation has been observed between grass species from the same genus. *F. rubra* had the highest foliar Si content and deposited more Si in damaged leaves than the other two species when plants were damaged under conditions of increased Si supply. However, it is the least abrasive species, presumably because its Si is deposited smoothly and evenly on the leaf surface and not in spines, and any phytoliths produced are few in number and, in contrast to spines, do not protrude substantially above the leaf surface. *D. cespitosa* has a very different strategy: a lower foliar Si content which was less affected by damage and Si addition, but what Si was present was deposited in numerous large spines, particularly at the tip, and under conditions of high Si supply, in a high density of additional structures which are absent under low Si supply. These structures may explain why this species has frequently been shown to be abrasive and unpalatable. Our results suggest that quantifying leaf Si concentration will not give a complete understanding of Sibased anti-herbivore defenses; rather examining how that foliar Si is deposited on the leaf surface will provide a better knowledge of how different plants use their Si and its likely impact on herbivores.

#### **ACKNOWLEDGMENTS**

We thank DLF-Trifolium for the *F. arundinacea* seeds, the horticultural staff at the University of York for assistance growing plants, and Meg Stark and Ian Wright for help with the SEM sample preparation and EDX spectroscopy. This work was funded by BBSRC training award to Sue E. Hartley (BB/K011715/1).

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 August 2014; accepted: 14 January 2015; published online: 11 February 2015.*

*Citation: Hartley SE, Fitt RN, McLarnon EL and Wade RN (2015) Defending the leaf surface: intra- and inter-specific differences in silicon deposition in grasses in response to damage and silicon supply. Front. Plant Sci. 6:35. doi: 10.3389/fpls.2015.00035*

*This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science.*

*Copyright © 2015 Hartley, Fitt, McLarnon and Wade. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Silicon, endophytes and secondary metabolites as grass defenses against mammalian herbivores

# *Otso Huitu1\*, Kristian M. Forbes1, Marjo Helander 2 , Riitta Julkunen-Tiitto3 , Xavier Lambin4 , Kari Saikkonen5 , Peter Stuart1 †, Sini Sulkama1 † and Sue Hartley6*

1 Suonenjoki Research Unit, Finnish Forest Research Institute, Suonenjoki, Finland

2 Section of Ecology, University of Turku, Turku, Finland

3 Department of Biology, University of Eastern Finland, Joensuu, Finland

4 School of Biological Sciences, University of Aberdeen, Aberdeen, UK

5 Plant Protection, Agrifood Research Finland, Jokioinen, Finland

6 Department of Biology, University of York, York, UK

#### *Edited by:*

Jane DeGabriel, University of Western Sydney, Australia

#### *Reviewed by:*

Daniel J. Ballhorn, Portland State University, USA Caroline Stolter, University of Hamburg, Germany

#### *\*Correspondence:*

Otso Huitu, Suonenjoki Research Unit, Finnish Forest Research Institute, Juntintie 154, FI-77600 Suonenjoki, Finland e-mail<EMAIL_ADDRESS>
#### *†Present address:*

Peter Stuart, Department of Botany and Zoology, Masaryk University, Brno, Czech Republic; Sini Sulkama, Department of Molecular Neurology, University of Helsinki, Finland

Grasses have been considered to primarily employ tolerance in lieu of defense in mitigating damage caused by herbivory.Yet a number of mechanisms have been identified in grasses, which may deter feeding by grazers. These include enhanced silicon uptake, hosting of toxin-producing endophytic fungi and induction of secondary metabolites. While these mechanisms have been individually studied, their synergistic responses to grazing, as well as their effects on grazers, are poorly known. A field experiment was carried out in 5 × 5 m outdoor enclosures to quantify phytochemical changes of either endophyteinfected (E+) or endophyte-free (E−) meadow fescue (Schedonorus pratensis) in response to medium intensity (corresponding with densities of ca. 1200 voles/ha for 5 weeks during 3 months) or heavy intensity (ca. 1200 voles/ha for 8 weeks during 3 months) grazing by a mammalian herbivore, the field vole (Microtus agrestis). A laboratory experiment was then conducted to evaluate the effects of endophyte infection status and grazing history of the grass diet on vole performance. As predicted, grazing increased foliar silicon content, by up to 13%. Grazing also increased foliar levels of phosphorous and several phenolic compounds, most notably those of the flavonols isorhamnetin-diglycoside and rhamnetin derivative. Silicon concentrations were consistently circa 16% higher in E+ grasses than in E-grasses, at all levels of grazing. Similarly, concentrations of chlorogenic acid derivative were found to be consistently higher in E+ than in E− grasses. Female voles maintained on heavily grazed grasses suffered higher mortality rates in the laboratory than female voles fed ungrazed grass, regardless of endophyte infection status. Our results conclusively demonstrate that, in addition to tolerance, grasses employ multi-tiered, effective defenses against mammalian grazers.

**Keywords: defense, endophytes, grasses, grazing, phenolics, secondary metabolites, silicon, voles**

# **INTRODUCTION**

Grasses are highly tolerant to grazing by means of their rapid regrowth capacity, basal meristems, underground storage organs and tillering capacity (Dyer et al., 1991; Karban and Baldwin, 2007). Indeed, grasses have been considered to primarily rely on these tolerance traits in lieu of physical or chemical defenses to mitigate damage caused by herbivory. However, a number of mechanisms have been identified in grasses that may deter feeding by grazers, and hence act as defenses (Vicari and Bazely, 1993). The most prominent of these is enhanced silicon (Si) uptake in response to damage (McNaughton and Tarrants, 1983; Massey et al., 2007a).

Following herbivore damage, Si is drawn from the soil and deposited systemically in the cell walls or lumina of new shoots as silica bodies or phytoliths (Ma and Yamaji, 2006; Currie and Perry, 2007). These forms of Si are extremely hard, and thereby increase the abrasiveness of leaf material (Massey and Hartley, 2006), potentially leading to accelerated tooth wear in grazers.

High Si levels also inhibit the absorption of nitrogen from digested plant material by mammalian herbivores (Massey and Hartley, 2006).

Grazers have often been observed to avoid plant material with high Si content (Gali-Muhtasib et al., 1992; Cotterill et al., 2007; Reynolds et al., 2012). However, the effects of Si on mammalian herbivore feeding behavior appear in many cases to be moderated or even over-ridden by a range of other factors including species identity (of both grass and herbivore), genotype or growing environment and other aspects of forage quality (Shewmaker et al., 1989; Massey et al., 2009; Soininen et al., 2013a).

Many grass species harbor systemic, vertically transmitted endophytic fungi, which may also increase the resistance of the host to herbivory (Clay, 1988; Cheplick and Clay, 1988; Saikkonen et al., 1998, 2006). Endophytic fungi are often producers of mycotoxins, which either reduce the palatability of the grass or render it toxic to the herbivore (Clay, 1988; Powell and Petroski, 1993; Siegel and Bush, 1996; Saikkonen et al., 1998, 2006). Both

the fungal load of grasses and the amount of toxins they produce may increase in response to herbivory, and hence act as induced defenses (Bazely et al., 1997).

While adverse effects of endophyte consumption are well documented for domestic livestock (reviewed by Clay, 1988), results from studies on wild mammalian grazers are scarce and ambiguous at best. Some studies report negative effects, such as decreases in population density (Coley et al., 1995), increased toxicity-induced mortality (Conover, 1998) and suppression of reproduction and growth (Jackson et al., 1996; Durham and Tannenbaum, 1998; Fortier et al., 2000; Conover, 2003), while others have found no effects at all (Barger and Tannenbaum, 1998; Saari et al., 2010). Thus, the effects of endophytic fungi on herbivores may be dosedependent and may vary between grass, fungi and herbivore species.

Grasses are known to produce an array of secondary metabolites, for example hydroxamic acids (Niemeyer, 1988), condensed tannins (Bernays et al., 1989), cyanogenic glycosides (Jones, 1998) and alkaloids, albeit at levels much lower than dicotyledons (Coughenour, 1985; Vicari and Bazely, 1993). Secondary metabolites found in grasses have been shown to have adverse effects on the performance of rodents when consumed in artificial diets (Lindroth and Batzli, 1984). However, only rarely have these compounds had measurable negative impacts on free-ranging mammalian herbivores at the concentrations in which they naturally occur in grasses. Exceptions again involve livestock: cyanogenic glycosides poisoning cattle (Georgiadis and McNaughton, 1988) and indole alkaloids reducing the palatability of grass for sheep (Simons and Marten, 1971; Marten et al., 1973).

Most studies have failed to identify significant effects of grazing on levels of defensive secondary metabolites in grasses (Lindroth and Batzli, 1986; Klemola et al., 2000). Nonetheless, graminivorous herbivores are highly selective feeders both within and between plant species (Freeland, 1974; Hjältén et al., 1996). As this behavior is to a great extent governed also by the secondary chemistry of their food plants (Jung and Batzli, 1981; Bergeron and Joudoin, 1989; Massey et al., 2007b), variation in grass secondary metabolite concentrations holds the potential to influence grazing mammals and their population demography (Freeland and Janzen, 1974).

Although these three potential defensive mechanisms of grasses – Si induction, endophytes, and secondary metabolites – have been individually studied, their synergistic responses to grazing and interactive effects on herbivores are poorly known. The aim of this study was to simultaneously quantify how levels of Si, other nutrients and secondary metabolite content interact in grasses in response to mammalian grazing and endophyte infection status. Furthermore, the aim was to evaluate the synergistic effect of these potential defense mechanisms on the physiological performance of a graminivorous herbivore. Although all of the grass defenses focussed on in this study are mechanisms through which herbivore populations may potentially be affected, the evidence for impacts of mechanisms in natural populations is missing.

Meadow fescue (*Scherodonus pratensis* ex. *Lolium pratense* and *Festuca pratensis*), which is commonly infected by the endophytic fungus *Epichloë uncinata* (Saari et al., 2009) was used as a model grass species. As an indicator of secondary metabolite production, we analyzed levels of phenolics, a widespread group of important defensive compounds (Rhoades, 1979) known to have adverse effects on graminivorous herbivores (Lindroth and Batzli, 1984). A common European grassland rodent, the field vole *Microtus agrestis* served as our model grazing herbivore.

The following specific hypotheses were tested: (1) vole grazing elevates levels of both Si and phenolics in grasses, (2) the magnitude of response of Si and phenolics to grazing differs between endophyte-infected and non-infected grasses, and (3) consumption of heavily grazed grass, as compared to consumption of less heavily grazed grasses, has negative impacts on vole physiological condition, and more so if the grass is endophyte-infected.

### **MATERIALS AND METHODS**

#### **STUDY SPECIES**

Meadow fescue is one of the most important native forage grasses in Nordic countries. It is also common outside of agronomic use in meadows, roadsides, and wastelands (Hämet-Ahti et al., 1998). Meadow fescue cultivars are often colonized by the systemic endophyte *E. uncinata*, which produces loline alkaloids, which appear to be non-toxic to large mammal herbivores (Clay and Schardl, 2002) but can be noxious to invertebrates and small vertebrates (Conover, 2003; Saikkonen et al., 2006; Huitu et al., 2008). A common meadow fescue cultivar "Kasper," registered and commercialized for use in Nordic countries in 1989 (Saari et al., 2009) was used for purposes of this study.

The field vole is a common and widespread small rodent species in Europe, including Fennoscandia (Myllymäki, 1977). It exhibits high-amplitude 3–4 year population cycles which are synchronous across large geographic areas (Sundell et al., 2004; Korpela et al., 2013). Field voles preferentially inhabit grassland habitats (Hansson, 1971; Myllymäki, 1977). Their diet consists predominately of grasses and forbs (Hansson, 1971).

#### **EXPERIMENTAL DESIGN AND PROTOCOL**

The study was conducted in fenced enclosures in Jokioinen, South–West Finland (60◦49 N, 23◦ 30 E) during summer 2011. The enclosures (20 in total in 5 × 4 configuration, each 25 m × 39 m; **Figure 1**) were established in May 2006 in an old agricultural field by tilling, fertilizing with cow manure (30 000 kg/ha) and sowing as monocultures either endophyte-free seed lots (E−; 0% endophyte frequency) or endophyte-infected seed lots (E+; 79% infection frequency) of the meadow fescue cultivar "Kasper" at a rate of 20 kg ha−1 (Saikkonen et al., 2013). In June 2007, plots were fertilized again with a commercial fertilizer [16:9:22 (N:P:K) with micronutrients, Kemira, product number: 0647334]. E+ and E− treatments were randomly assigned to 10 enclosure pairs. Seed lots were obtained from seed production farms via the Finnish Food Safety Authority (EVIRA), Seed Certification Unit, Loimaa, Finland. Four years after establishment, the cover percentages of meadow fescue had decreased from 100 to 75% and 98% in E– and E+ plots, respectively, due to weed invasion (Saikkonen et al., 2013).

Each enclosure was constructed of a sheet metal fence (embedded 60 cm below ground while 60 cm remained above ground) in

**FIGURE 1 | Aerial photo of experimental enclosure complex.** The denotation E+ indicates those 25 × 39 m enclosures which were vegetated by endophyte-infected meadow fescue, and E− those with endophyte-free meadow fescue. The denotation G+ in the smaller plots pictured in the inset indicate those plots in which field voles grazed through the summer, and G− those, which were not grazed. Photo courtesy of the National Land Survey of Finland.

order to retain the experimental voles and exclude voles of natural populations and small mammal predators.

Every second enclosure pair of the 10 available pairs was selected for the experiment (**Figure 1**). Four 5m × 5 m sheet metal plots arranged in 2 × 2 squares were constructed (**Figure 1**) in one randomly assigned corner (corners bordering the outside of the enclosure complex were excluded to reduce predator attraction) of each of the 10 enclosures (5 × E+ and 5 × E−). Two plots per enclosure were randomly designated as vole grazing plots (G+) and two as non-grazed control plots (G−).

In total, the experiment consisted of 10 replicates of each of the following factorial treatments: vole grazing in endophyte-infected plots (G+E+), vole grazing in endophyte-free plots (G+E−), no grazing in endophyte-infected plots (G−E+), and no grazing in endophyte-free plots (G−E−). The grazing treatment was begun in late May 2011 by introducing two adult field voles, trapped in the vicinity of the enclosures and in Suonenjoki, Eastern Finland, into each G+ plot. Prior to introduction, all voles were housed in standard laboratory rodent cages (43cm × 26cm × 15 cm; Tecniplast, Italy) with ad libitum water, feed pellets (Altromin 1314F; Altromin Spezialfutter GmbH and Co. Germany), and turnip.

Two levels of grazing intensity we applied to ensure adequate responses of grasses to the grazing treatment. The medium grazing intensity treatment involved two voles grazing in each plot continuously for 2 weeks, and thereafter four voles grazing for 3 weeks, either directly after the first 2 weeks, or after a 3 week period of no

grazing. The heavy grazing intensity treatment involved two voles grazing in each plot continuously for 2 weeks, and thereafter four voles grazing first for 3 weeks, followed by 3 weeks of no grazing and finally another 3 weeks of grazing by four voles.

In summary, the medium grazing intensity plots received 14 days of grazing at a vole density corresponding to 800 voles / ha (2 voles × 0.0025 ha = 800 voles/ha) plus 21 days of grazing at a vole density corresponding with 1600 voles/ha (4 voles × 0.0025 ha = 1600 voles/ha). The high intensity grazing treatments received 14 days of grazing at 800 voles / ha plus 42 days of grazing at 1600 voles/ha. The collective grazing intensity over the experiment approximated 1200 voles/ha for 5 weeks during 3 months and ca. 1200 voles / ha for 8 weeks during 3 months for the medium and heavy grazing intensity treatments, respectively. The level of grazing on individual grasses was not measured, but visual inspection indicated that while heavy grazing did have a substantial effect on vegetation, grasses were at no point during the experiment entirely deplete within the plots.

Live trapping was conducted in mid-June in all enclosures to measure the body mass of voles in the G+ enclosures and to verify that no voles had entered the G− enclosures. As expected, no voles were encountered in the G− enclosures. However, only circa 25% of the originally introduced voles were trapped in the G+ enclosures over three nights. This strongly indicated predation within the experimental enclosures, and a cat was occasionally seen around the enclosure complex.

Therefore, the experimental grazing protocol was altered from its original form. In mid-June, all voles were removed from the plots. Four field voles were then introduced into half of the G+ plots for a period of 3 weeks. Thereafter, the voles were trapped and translocated to the remaining plots. This was again repeated after 3 weeks, with four voles returned to the plots that were first grazed. During this 9 week period, only seven voles disappeared, and those that did were replaced upon translocation. All voles were live trapped out of the plots at the end of August.

#### **SAMPLE COLLECTION AND PHYTOCHEMICAL ANALYSES**

To evaluate the effects of grazing on phytochemistry, meadow fescue samples were collected in each of the plots three times during the summer: immediately prior to introduction of voles, once at the beginning of August and once at the beginning of September (1 week after voles had been removed). Each time, one tiller was removed at the base from three randomly selected meadow fescue plants per plot and pooled into one sample. During the first sampling occasion, samples from the two G+ and two G− plots per enclosure were also pooled.

The Si, phosphorous (P), nitrogen (N), and carbon (C) contents of the samples were analyzed for each of the three sampling occasions. For these analyses, grass samples were washed under water, oven dried at 80◦C and ground into a fine, non-fibrous powder. Three hundred milligram of this powder was pressed in a hydraulic press into a 3 mm thick, 13 mm diameter pellet with a pressure of 13 bar. Si and P content (expressed as percentage dry weight) was analyzed from the pellets with a portable X-ray fluorescence spectrometer (Niton XL3t900 GOLDD Analyzer; Thermo Scientific,Winchester, UK), as described by Reidinger et al. (2012). As this technique is non-destructible, the same pellets were used for the analysis of N and C. This was done by ISO 13878 and ISO 10694 based methods, respectively, using a LECO/CHN analyser (Leco Co., USA).

Phenolic compounds were analyzed only from samples collected on the last occasion, after the grazing treatment. These samples were air dried at room temperature for 1 week prior to analyses. All soluble non-tannin phenolic compounds were extracted and HPLC-analyzed from the grass leaf samples according to the protocol described in Nybakken et al. (2012).

#### **VOLE LABORATORY EXPERIMENT**

At the beginning of September, a laboratory experiment was carried out to evaluate how earlier grazing and endophyte infection status affected vole physiological condition and performance. 96 wild field voles, captured near the towns of Suonenjoki, central Finland (62◦ N, 27◦ E) and Jokioinen, were housed in same sex pairs in a decommissioned greenhouse, in standard laboratory cages containing peat and straw as bedding and nest material. The voles were assigned to one of four food treatment groups (*n* = 24 voles per treatment group), which received *ad libitum* grass collected daily from the experimental plots (treatments: E+G+, E+G−, E−G+, and E−G−). For this segment of the study, all grass collected from grazed enclosures was pooled and not distinguished between heavy or medium grazing. Voles also received pieces of potato for extra hydration.

Experimental feeding began on September 10th and continued until the conclusion of the experiment on September 26th. Throughout this period, voles were checked daily for survival, and each animal was weighed using a spring balance (Pesola AG, Switzerland; accuracy ± 1 g) every 3–4 days (six measurement occasions in total). In the beginning and at the end of the feeding period, a blood sample was collected from the retro-orbital sinus of each vole using a capillary tube. Tubes were then centrifuged at 12000 g for five minutes, and haematocrit expressed as the percentage of packed red blood cells in total volume. The feeding experiment was terminated and all voles euthanized due to an increased rate of mortality in the end of the second experimental week, for reasons that remain unidentified.

The experiment was carried out under permit from the Animal Ethics Council of the State Provincial Office of Southern Finland.

#### **STATISTICAL ANALYSES**

Variation in grass Si, P, N, and C content was analyzed with linear mixed models using the maximum likelihood method, where each of the elements was in turn the response variable. Grazing intensity (heavy, medium or control), endophyte infection status (E+ or E−), time (June, August, September) and all interactions were initially entered in the models as class explanatory variables. Time was designated a repeated factor, with plot as the subject. In all cases, AIC-based model selection (Burnham and Anderson, 2000) indicated unstructured as the most parsimonious covariance type. Enclosure was entered as a random factor.

Variation in phenolics concentrations at the end of summer was also analyzed with separate linear mixed models for each compound using the maximum likelihood method. Initially, all peak intensity values for the different compounds were loge transformed. Grazing intensity, endophyte infection status and their interaction were entered as fixed explanatory variables, while enclosure and plot (nested within enclosure) were designated random factors.

The overall response of meadow fescue to the treatments in terms of their phenolic content was also analyzed with multivariate analysis of variance using all 16 identified phenolic compounds as response variables, and grazing intensity, endophyte infection status and their interaction as explanatory variables.

Changes in the body mass and haematocrit value of voles that survived to the end of the laboratory experiment were analyzed with linear mixed models, with the percentage of change relative to the beginning of the experiment as the response variable. Grazing history (grazed or not) and endophyte infection status of the grass they were fed, vole sex and body mass at the beginning of the experiment, together with all possible three-way interactions, were entered as fixed explanatory variables. Identity of the cage in which the voles were housed was entered as a random factor. Full models were reduced by removing explanatory terms on the basis of AIC. Model fit was assessed by visual observation of the final model residuals.

Vole survival during the laboratory experiment was analyzed with generalized linear mixed models, using vole survival as a binary response variable with a logit link. Grazing history and endophyte infection status of the grass, vole sex, and all interactions, were entered as fixed explanatory variables and vole cage as a random factor.

Full models were reduced in all analyses by removing explanatory terms on the basis of AIC – terms were removed if this reduced the AIC value by >2 units (Burnham and Anderson, 2000). Model fit was assessed in the linear mixed models by visual inspection of the final model residuals, and by the ratio of the generalized chi-square statistic and its degrees of freedom in the generalized linear mixed model. All analyses were carried out with SAS statistical software v9.3 (SAS® Institute Inc., Cary, NC, USA).

# **RESULTS**

Grazing elevated the Si content of grasses (*F*2,24.4 = 3.50, *p* = 0.046). Highest levels were recorded in the heavy grazing treatment (mean ± SE. Si content as percentage dry weight = 1.56 ± 0.07%), followed by medium grazing (1.54 ± 0.08%), while control grasses had the lowest values (1.36 ± 0.05%; **Figure 2**). Si levels were consistently higher in E+ grasses than in E− grasses, with no significant interactive effect of grazing (E+: mean ± SE. Si content as percentage dry weight = 1.60 ± 0.06%; E− = 1.38 ± 0.06%; **Figure 2**).

Likewise, grazing elevated the P content of grasses (*F*2,28.4 = 3.78, *p* = 0.035). Highest levels were recorded in the medium grazing treatment (mean ± SE. P content as percentage dry weight = 0.42 ± 0.01%), followed by heavy grazing (0.42 ± 0.01%), while control grasses had the lowest values (0.40 ± 0.01%; *t* control vs. heavy24.8 = −2.03, *p* = 0.054; *t* control vs. medium 31.2 = −2.43, *p* = 0.021; *t*heavy vs. medium30.5 = −0.35, *p* = 0.73). The level of P declined in E+ grasses over the summer, whereas that in E− grasses remained stable (*F*2,27.6 = 3.79,

*p* = 0.035). No differences were found in concentrations of N, C, or in C/N-ratio relative to grazing intensity, endophyte infection status, time or their interactions (all *p*-values > 0.08).

A two-way multivariate analysis of variance revealed a significant interaction between grazing intensity and endophyte infection status on the phenolics profile of the grasses (Wilks' Lambda = 0.12, *F*32,38 = 2.27, *p* = 0.008). One-way multivariate tests with either E+or E−samples did not clearly indicate which of the main effects had a stronger effect on grass phytochemistry. The varying degrees of grazing intensity tended to influence the phenolics profile of endophyte-free samples (Wilks' Lambda = 0.001, *F*32,4 = 4.58, *p* = 0.074), but these differences were less pronounced in endophyte-infected samples (Wilks' Lambda = 0.02, *F*32,4 = 2.40, *p* = 0.21).

Five of the 16 analyzed phenolic compounds exhibited an elevated response to grazing – chlorogenic acid, isorhamnetindiglycoside, myricetin-glycoside, isorhamnetin 3-glucoside, and rhamnetin derivative (**Table 1**). Concentrations of chlorogenic acid derivative were higher in E+ than in E− grasses. Two compounds were affected by the interaction between grazing intensity and endophyte status. Levels of quercitrin, in particular, were positively associated with grazing intensity, but only in E+ grasses (**Table 1**).

The body mass of voles maintained in the laboratory on endophyte-infected and endophyte-free grasses responded differently to the grazing history of their diet. Voles feeding on endophyte-free grass that had been grazed throughout the summer lost relatively more mass than voles in the other three groups (mean ± SE. mass change during experiment as percentage: E−G−=+3.90 ± 3.12%, E−G+=−8.92 ± 3.08%, E+G−=+0.74 ± 2.88%, E+G+=+0.62 ± 3.18%). Feeding on previously grazed grass also had a significant negative main effect on vole body mass change (mean ± SE. mass change: G−=+ 2.32 ± 2.15%, G+=−4.15 ± 2.18%). Females lost more body mass than males, irrespective of dietary group (mean ± SE.

mass change: males = +3.21 ± 2.26, females = −5.04 ± 2.06%; **Figure 3**).

Females that had fed on endophyte-infected grass had elevated haematocrit levels compared to those fed on endophyte-free grasses (mean ± SE.: E+ = 51.3 ± 0.8, E− = 48.9 ± 0.9; *F*1,19.7 = 4.55, *p* = 0.046). Conversely, females maintained on heavily grazed grasses suffered ca. 45% higher mortality rates than females fed ungrazed grass, regardless of endophyte infection status; these differences did not manifest in males (**Figure 4**).

#### **DISCUSSION**

In support of our hypotheses, vole grazing elevated levels of both Si and phenolics in grasses. Endophyte infection alone also elevated grass Si content, to an extent comparable to that of grazing. However, changes in the levels of Si in response to grazing did not differ between E+ and E− grasses. Grazing and endophyte infection status did interact in altering the phenolic compound profile of grasses. Also in support of our hypothesis, consumption of heavily grazed grass had negative impacts on vole physiological condition, but these effects were conditional on endophyte infection status. Although endophyte infection elevates Si content as much as grazing alone does, our results overall suggest that grazing has a more prominent effect on grass defenses than endophyte infection status.

#### **GRASS RESPONSES TO GRAZING**

As predicted, grazing-induced the uptake of Si from the soil into the grass. This response has been documented several times in association with various grazer taxa (McNaughton and Tarrants, 1983; Massey and Hartley, 2006; Massey et al., 2007a), and the biochemical processes underlying it are well known (Ma and Yamaji, 2006; Currie and Perry, 2007). Si accumulation has been identified as the most plausible, and potent defense mechanism of grasses against graminivorous voles (Massey and Hartley, 2006). It has even been suggested as a candidate factor for generating multiannual population cycles in rodents, as its induction in response to grazing may occur with a delay relative to rodent density, and the induced levels of Si persist in the grasses for sufficiently long (Massey et al., 2008; Reynolds et al., 2012).

Although the accumulation of Si has in many study systems received support as a defense mechanism against grazers, many others have failed to do so. For example, Shewmaker et al. (1989) could not demonstrate any effects of Si content on the grazing preference of sheep, which appear to be less responsive to variations in grass Si content than small rodents (Massey et al., 2009). Densities of African grazing ungulates have been shown to be either negatively (McNaughton and Tarrants, 1983) or positively (Georgiadis and McNaughton, 1990) associated with the Si content of their food plants. This contingency in responses is likely to reflect differences in grazing levels and hence in the magnitude of Si induction, given the known impacts of both frequency and intensity of damage on the Si response (Massey et al., 2007a; Reynolds et al., 2012), as well as variations in the range of plant, herbivore and environmental factors influencing plant-herbivore interactions (e.g., Soininen et al., 2013a).

In subarctic Fennoscandia, where vole densities are 2–3 orders of magnitude lower than simulated in this experiment, grass Si

**Table 1 | Mean peak intensity values of the phenolic compounds analyzed, grouped by grazing intensity, and endophyte status of the grasses.**

Significant effect denotes which of these terms was significant at a risk level of α = 0.05 (borderline values indicated in column Direction). Direction denotes pairwise differences between levels of the significant effect and their direction; C, M, and H denote control, medium and heavy grazing intensities, E+ and E− indicate endophyte-infected and non-infected grasses, respectively.

levels appear to be influenced more by grass species, their genotype or their growing environment than by the mostly low grazing pressure (Soininen et al., 2013a). Indeed, the majority of experimental studies in which Si has become induced in response to rodent grazing have been conducted in laboratory or greenhouse conditions (Massey and Hartley, 2006; Massey et al., 2007a; Reynolds et al., 2012, but see Massey et al., 2008), or as is the case here, in small outdoor enclosures. In these experimental conditions it is relatively easy to impose high damage levels in terms of biomass removed (Reynolds et al., 2012) or high vole densities (this study). It seems likely that both vole densities and the frequency and intensity of plant damage are likely to be lower in natural systems, which are also subject to a greater range of environmental variability (including climatic factors, soil type, plant genotypes, and growth stage), making the detection of Si induction in the field at the landscape scale more challenging.

While no differences were found in concentrations of N, C, or in C/N-ratio relative to grazing intensity or endophyte infection status, grazing did elevate the P content of grasses. Levels of phosphorous have been found to increase in response to grazing in grasses, but this primarily occurs to facilitate shoot regrowth after defoliation, rather than to act as an induced defense against grazers (Chapin and Slack, 1979; Donaldson et al., 1984; McNaughton

and Chapin, 1985). It is evident that the primary chemistry of the grasses did not exhibit pronounced changes in response to defoliation by grazing.

Grazing increased the content of five of the 16 analyzed phenolic compounds – chlorogenic acid and five flavonols, isorhamnetin-diglycoside, myricetin-glycoside, isorhamnetin 3 glucoside, and rhamnetin derivative (**Table 1**). Many phenolics are inducible defensive compounds, which either inhibit digestion by herbivores, are deterrent or even directly toxic (Lindroth and Batzli, 1984; Karban and Baldwin, 2007). Their concentrations in grasses are generally low relative to other plant taxa (Bernays et al., 1989) and their collective levels appear not to respond greatly to grazing, at least by rodents (Lindroth and Batzli, 1986). However, in response to herbivory, the relative concentrations of different individual phenolic compounds may change dramatically without changing total concentrations, and it has been demonstrated that individual compounds can impact adversely on voles (Lindroth and Batzli, 1984).

A clear caveat of our study on grass phytochemical responses to grazing is the fact that concentrations of all possible defensive compounds, such as cyanogenic glycosides, hydroxamic acids, or alkaloids were not comprehensively analyzed. A number of these have been found to respond to grazing and to have adverse effects

**FIGURE 3 | Changes in body mass of voles maintained in the laboratory on previously grazed or ungrazed endophyte-infected (E+) or endophyte-free (E−) grass; (A) males, (B) females.** Differences of changes in body mass of voles relative to the treatments and vole sex: Fendophyte × grazing1,40.9 = 4.14, p = 0.049; Fgrazing1,38.8 = 4.38, p = 0.043; Fsex1,40.3 = 7.20, p = 0.01.

on herbivores (reviewed byVicari and Bazely, 1993). The nature of these associations in our study system remains to be investigated in future research.

#### **EFFECTS OF ENDOPHYTE INFECTION ON GRASS PHYTOCHEMISTRY**

Si levels were found to be to be slightly higher in E+ grasses than in E− grasses. Si has been shown to be positively associated with drought resistance in grasses, as it forms physical structures on leaves which reduce transpiration through stomata (Lux et al., 2002). Si is also known for its capacity to inhibit the growth of pathogenic fungi (Fauteux et al., 2005) and although often described as having mutualistic rather than antagonistic interactions with its host plants (Saikkonen et al., 2006), it is plausible that endophytic fungi are in some cases identified by their hosts as harmful pathogens (Saikkonen et al., 1998, 2004). An alternative explanation for the elevated Si levels observed in E+ grasses in this study may therefore be an evolutionarily conserved defensive response against the intrusion of a foreign fungal organism, even potentially mutualistic ones.

Similarly, chlorogenic acid derivative levels were higher in E+ than in E− grasses. Chlorogenic acid has been shown to act as a chemical defense against invertebrate herbivores (Bernays et al., 2000; Ikonen et al., 2001), though very little is known of how, or if, chlorogenic acid acts as a defense against fungal infections in plants, as Si does.

#### **INTERACTIVE EFFECTS OF GRAZING AND ENDOPHYTE INFECTION ON GRASS PHYTOCHEMISTRY**

Two phenolic compounds were affected by the interaction between grazing intensity and endophyte status. Levels of quercitrin (also known as quercetin 3-rhamnoside), in particular, were positively associated with grazing intensity, but only in E+ grasses (**Table 1**). Quercitrin consumption has been shown to reduce the growth of weanling voles, and most profoundly so when dietary protein levels are low (Lindroth and Batzli, 1984). The effects of quercitrin on voles appear to be toxic, as dietary protein levels have been shown not to affect protein digestibility – rather, low dietary protein levels appear to reduce the capacity of voles to detoxify quercitrin (Lindroth and Batzli, 1984).

The grazing-induced elevation of Si may have more farreaching effects than the up-regulation of the physical defense of plants. For example, Si appears also to have a functional role in the systemic response of plants to pathogens, through the activation of general defensive pathways, e.g., involving salicylic, or jasmonic acids (Fawe et al., 1998; Rodrigues et al., 2004; Fauteux et al., 2005). Elevated Si levels have also been shown to increase the accumulation of lignin, phenolic compounds, chitinases, and peroxidases (Fawe et al., 1998). As such, Si is likely to be integral in the regulation of terpene or phenolic –based defenses in stressed plants (Fauteux et al., 2005).

A caveat of our study is that we were unable to determine the effects of vole grazing on either the amount of endophyte hyphae per grass leaf or the concentrations of mycotoxin alkaloids that the fungus produces. Bazely et al. (1997) demonstrated that intensive sheep grazing increased hyphae loads and/or alkaloid production. A similar mechanism may occur with vole grazing, though this too remains a topic for further investigation.

#### **EFFECTS OF DIET QUALITY VARIATION ON VOLE CONDITION**

Voles that consumed previously grazed grass lost body mass in the laboratory. Massey and Hartley (2006) established that an elevated concentration of Si in the diet is detrimental to graminivorous voles, in that it reduces their capacity to absorb nitrogen from their diet, leading to reduced growth rates in juveniles and

adult females, and our study confirms this. Conversely, there are far fewer cases in which secondary metabolites have been shown to have similar negative effects on graminivorous mammals (see Simons and Marten, 1971; Marten et al., 1973; Georgiadis and McNaughton, 1988), let alone voles. Although grasses produce a wide array of secondary metabolites (reviewed by Vicari and Bazely, 1993), their effects on rodents appear to manifest only when animals are fed on experimental diets in laboratory conditions (e.g., Lindroth and Batzli, 1984). In light of this, it seems more parsimonious to attribute the observed body mass loss of voles to elevated Si content in the grasses than phenolics content. However, our experimental design does not enable us to firmly discriminate between these factors.

Females, in particular, suffered mass loss while maintained in the laboratory, regardless of treatment. The voles were offered only one species of grass as diet. Consumption of such a monotonous diet is highly unlikely for voles in natural habitats (Myllymäki, 1977; Soininen et al., 2013b). The fact that females suffered greater mass loss than males may be due to intersexual differences in dietary requirements related to, e.g., reproduction (Barboza and Bowyer, 2000; of note, none of the females in our experiment were gravid). Unfortunately, virtually no information exists on sex differences in dietary intake in rodents.

Females that had fed on endophyte-infected grass had elevated haematocrit levels compared to those fed on endophyte-free grasses. Haematocrit measures the relative volume of red blood cells in whole blood, and its values may increase due to either proliferation of red blood cells, or, more commonly, due to a reduction in blood plasma content due to dehydration (Fair et al., 2007). A dehydration-induced reduction in haematocrit often occurs in association with prolonged fasting in animals (Vleck et al., 2000). It is likely that the ultimate factors which lead to female mass loss and high death rate also contributed proximately to their haematocrit levels. Thereby, it is plausible that females were more sensitive either to the increased Si content of the E+ grasses, or to the endophyte itself, than were male voles.

Highest levels of mortality were recorded for females maintained on grazed grasses. Conversely, endophyte infection status did not affect mortality rates. Although endophyte infection status appeared to be associated with female food avoidance in the laboratory, as judged by hematocrit levels, these effects were not pronounced enough to impact survival. Collectively, our results indicate that grazing-induced changes in the quality of diet, i.e., increased Si and phenolics content, outweigh the potential negative effects of endophyte infection on the physiological condition of voles (see Lindroth and Batzli, 1984; Massey and Hartley, 2006). Furthermore, we demonstrate here that heavy and prolonged grazing may reduce the quality of graminivorous vole diet to the extent that it has negative effects on vole survival. By extension, as the most profound negative impacts were observed in females, these effects hold the potential to carry over to population growth. However, we acknowledge that the artificial nature of our laboratory may have generated spurious associations between food quality and vole performance. More detailed experimental research is therefore still called for to elucidate these causal relationships in entirely natural surroundings.

# **CONCLUSION**

Our results render support to the hypothesis that food quality may indeed have a limiting effect on vole population growth, in cases when grazing is severe and long enough (see Reynolds et al., 2012). We conclusively demonstrate here that grasses are capable of employing multi-tiered, effective defenses against mammalian grazers. We do not imply that these findings are universally applicable to all grazing ecosystems – rather, we highlight the need for more detailed investigations on the entire metabolome and the relative effects of defense mechanisms thus far identified in grasses to elucidate the role of food quality as a determinant of herbivore population dynamics.

## **ACKNOWLEDGMENTS**

This study was supported by the Academy of Finland (grant no. 133495 to Otso Huitu; grants no. 137909 and 110658 to Kari Saikkonen) and by the NERC (grant no. NE/F003994/1 to Xavier Lambin). We thank Dr. Stefan Reidinger and Dr. James Stockdale for help with the silicon analyses. Technician Sinikka Sorsa conducted the phenolic extractions. Stephen Ryan and Anaïs Zimmer assisted with field work.

# **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 May 2014; accepted: 30 August 2014; published online: 17 September 2014.*

*Citation: Huitu O, Forbes KM, Helander M, Julkunen-Tiitto R, Lambin X, Saikkonen K, Stuart P, Sulkama S and Hartley S (2014) Silicon, endophytes and secondary metabolites as grass defenses against mammalian herbivores. Front. Plant Sci. 5:478. doi: 10.3389/fpls.2014.00478*

*This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Huitu, Forbes, Helander, Julkunen-Tiitto, Lambin, Saikkonen, Stuart, Sulkama and Hartley. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Silica uptake by *Spartina*—evidence of multiple modes of accumulation from salt marshes around the world

#### *Joanna C. Carey1 \* and Robinson W. Fulweiler 1,2*

*1 Department of Earth and Environment, Boston University, Boston, MA, USA 2 Department of Biology, Boston University, Boston, MA, USA*

#### *Edited by:*

*Julia Cooke, University of Western Sydney, Australia*

#### *Reviewed by:*

*Heidi Renninger, Boston University, USA*

*Cathleen Wigand, US EPA, USA*

#### *\*Correspondence:*

*Joanna C. Carey, United States Environmental Protection Agency, 27 Tarzwell Drive, Narragansett, RI 02882, USA e-mail<EMAIL_ADDRESS>
Silicon (Si) plays a critical role in plant functional ecology, protecting plants from multiple environmental stressors. While all terrestrial plants contain some Si, wetland grasses are frequently found to have the highest concentrations, although the mechanisms driving Si accumulation in wetland grasses remain in large part uncertain. For example, active Si accumulation is often assumed to be responsible for elevated Si concentrations found in wetland grasses. However, life stage and differences in Si availability in the surrounding environment also appear to be important variables controlling the Si concentrations of wetland grasses. Here we used original data from five North American salt marshes, as well as all known published literature values, to examine the primary drivers of Si accumulation in *Spartina*, a genus of prolific salt marsh grasses found worldwide. We found evidence of multiple modes of Si accumulation in *Spartina*, with passive accumulation observed in non-degraded marshes where *Spartina* was native, while rejective accumulation was found in regions where *Spartina* was invasive. Evidence of active accumulation was found in only one marsh where *Spartina* was native, but was also subjected to nutrient over-enrichment. We developed a conceptual model which hypothesizes that the mode of Si uptake by *Spartina* is dependent on local environmental factors and genetic origin, supporting the idea that plant species should be placed along a spectrum of Si accumulation. We hypothesize that *Spartina* exhibits previously unrecognized phenotypic plasticity with regard to Si accumulation, allowing these plants to respond to changes in marsh condition. These results provide new insight regarding how salt marsh ecosystems regulate Si exchange at the land-sea interface.

**Keywords:** *Spartina***, salt marsh, silica, accumulation, grasses, wetland, ecosystem service**

# **INTRODUCTION**

The role of nitrogen (N) and phosphorus (P) in plant productivity is well recognized by plant ecologists. Less well constrained is the importance of silicon (Si) in plant growth, maintenance, and fitness (Cooke and Leishman, 2011). In terrestrial ecosystems, Si is classified as a "quasi-essential" nutrient (Epstein, 2009) because it protects plants from a variety of stressors, including desiccation, heavy metal toxicity, and predation (Epstein, 1994; Hodson and Evans, 1995; Liang et al., 2007). Through their roots, plants take up dissolved silica (DSi) (SiO2) from the porewater, or soil solution, and transport it through the transpiration stream via the xylem. As water leaves the plant, Si becomes concentrated at transpiration termini and is deposited as biogenic Si (BSi). As such, plants often demonstrate increasing BSi concentrations with age or growth (Jones and Handreck, 1967; Epstein, 1994), due to continual precipitation of BSi at transpiration termini and the inability of plants to translocate Si once precipitated (Raven, 1983; Epstein, 1994). Although the majority of Si in plants is located at transpiration termini (e.g., leaves), Si is also found throughout other portions of plants, such as the roots, rhizomes, and stems (Struyf et al., 2005b; Querné et al., 2012; Carey and Fulweiler, 2013). The concentration of Si in plants is often higher than many macronutrients such as N and potassium (K). Si concentrations typically range from 0.1 to 10% by weight (by wt.) (Ma et al., 2001), the largest range of any element found in plants (Epstein, 1994).

The accumulation of Si in plants occurs via three possible modes of uptake: (1) active accumulation, where plants take up more Si than they would through water uptake alone, (2) passive accumulation, where plants have similar Si and water uptake rates, and (3) rejective or excluder accumulation, where Si uptake is slower than water uptake (Raven, 1983; Takahashi et al., 1990; Ma et al., 2001). These three possible modes of Si accumulation can be determined in several ways (Jones and Handreck, 1967; Takahashi et al., 1990; Ma et al., 2001). First, Si accumulation can be defined by the concentrations of Si in the aboveground plant tissue alone, where active accumulators typically have a dry weight of SiO2 *>*1% by wt., passive accumulators between 0.5 and 1% by wt., and excluders *<*0.5% by wt. (Ma et al., 2001; Street-Perrott and Barker, 2008; Hou et al., 2010; Carey and Fulweiler, 2012). The ratio of Si to calcium (Ca) is another means of determining the mode of Si accumulation, with Si:Ca ratios *>*1 indicating active accumulation, 0.5–1.0 indicating passive accumulation, and *<*0.5 indicating Si exclusion (Takahashi et al., 1990; Ma et al., 2001; Ma and Takahashi, 2002). Finally, the mode of Si accumulation can be determined based on the relationship between DSi porewater concentrations and aboveground biomass BSi concentrations (Jones and Handreck, 1967; De Bakker et al., 1999; Norris and Hackney, 1999; Ma et al., 2001). In this case, a positive relationship between porewater DSi concentrations and above ground BSi concentrations indicates passive accumulation, a negative relationship indicates active accumulation, and no relationship indicates rejective accumulation (Raven, 1983; De Bakker et al., 1999).

While all vegetation contains some Si (Epstein, 1994), grasses (*Poaceae*) and sedges (*Cyperaceae*) typically accumulate the most (Jones and Handreck, 1967; Raven, 1983; Ma and Takahashi, 2002), which has been attributed to active Si accumulation by these plants (Jones and Handreck, 1967; Raven, 1983). However, it remains uncertain whether all wetland grasses fall into this category of active accumulation. For example, evidence exists that *Spartina* grasses, which are one of the most prolific genus of salt marsh grasses worldwide, are passive accumulators (Hou et al., 2010; Querné et al., 2012) and possibly even rejector plants (De Bakker et al., 1999).

Understanding the mechanism responsible for Si accumulation by salt marsh plants is important for understanding the controls on Si exchange at the land-sea interface. Tidal marshes, are large reservoirs of Si (Struyf et al., 2005b; Carey and Fulweiler, 2013), and have been shown to play a critical role in regulating Si availability in adjacent estuarine waters (Struyf et al., 2005a; Jacobs et al., 2008; Vieillard et al., 2011). This has important consequences for marine trophic structure (Officer and Ryther, 1980), as diatoms, a dominant type of phytoplankton in temperate coastal waters, require as much Si as N on a molar basis to survive (Redfield et al., 1963). Because the Si found in marsh plants (i.e., BSi) dissolves several orders of magnitude faster than mineral silicates (Alexandre et al., 1997; Cornelis et al., 2010a), understanding how marsh grasses sequester Si is a key step for understanding how salt marshes regulate Si exchange in these dynamic ecotones.

Currently, controls on Si accumulation in salt marsh grasses are not well understood. For example, in addition to uncertainties regarding the mode of Si accumulation (i.e., active, passive, or rejective accumulation), the role of plant age or growth rate in controlling Si accumulation remains unclear (Querné et al., 2012). Although increasing BSi content with growth has been observed for *Spartina* (De Bakker et al., 1999; Norris and Hackney, 1999; Querné et al., 2012; Carey and Fulweiler, 2013) and several species of freshwater marsh plants (Struyf et al., 2005b), Hou et al. (2010) found no increase in *S. alterniflora* BSi concentrations over the growing season. The highly dynamic nature of salt marshes, which experience a wide range in nutrient availability, sediment type, and hydrologic conditions, could be responsible for the non-uniform BSi concentrations found in salt marsh plants. Furthermore, the amount of Si in the surrounding environment may also exert an important control over plant Si accumulation, especially in cases of passive Si accumulation (Struyf et al., 2005b). In these cases, increased Si availability in soil solution, or porewater, would result in higher Si accumulation in the plants.

The primary objective of this study was to determine the drivers of BSi accumulation in *Spartina*, one of the most common types of salt marsh grasses found worldwide. We hypothesize that differences in *Spartina* Si accumulation are driven by environmental conditions and plant origin (native vs. non-native). To test this hypothesis, we used original data from five New England (USA) salt marshes, as well as published literature values, to examine the mode of Si accumulation, and the role of plant growth and *in situ* Si availability (in sediment and porewater) in controlling BSi accumulation in *Spartina*. We then developed a conceptual model which suggests that differences in aboveground BSi concentrations are due not only to differences in Si availability in marshes, but also different modes of Si uptake, representing a previously unrecognized form of phenotypic plasticity in these plants.

#### **MATERIALS AND METHODS**

We collected samples for Si accumulation at the height of growing season (spring) and during peak biomass (summer) in five marshes in New England, USA: a salt marsh in northern Maine (Site 1) and four Rhode Island (RI) salt marshes (Sites 2–5) (**Figure 1**, **Table 1**). All of the marshes displayed patterns of vegetation that are typical in New England marshes (Bricker-Urso et al., 1989; Wigand and Roman, 2012), with areas of "low marsh" that are inundated with tides twice a day consisting entirely of *Spartina alterniflora* grasses, while the areas of "high marsh" are inundated less frequently and are dominated by *S. patens* vegetation. Site 1 is a relatively undisturbed fringing salt marsh, located adjacent to Acadia National Park, draining an undeveloped watershed. The RI marshes cover a range of anthropogenic nutrient loadings and salinities (**Table 1**). Three of these marshes (Site 2–4) span the length of Narragansett Bay, from high to low nutrient inputs (DSi, N, P) (**Figure 1**, **Table 1**). Site 2 is a back-barrier marsh located on the east side of the Providence River Estuary, Site 3 is back-barrier marsh located on Prudence Island within the Narragansett Bay National Estuarine Research Reserve, and Site 4 is a fringing marsh located on the south side of Zeek's

**FIGURE 1 | Map of study region in New England, USA.**

*Unless otherwise noted, all values summer averages* ± *standard error.*

∗*Does not include NH4*+ *,* ∗∗*annual average.*

Creek in Jamestown, RI (**Table 1**). Site 5 is fringing marsh in Great Salt Pond located 21 km south of the coast of RI, on a relatively undisturbed island (Block Island) exposed to low-nutrient ocean water.

We measured net Si accumulation in the aboveground vegetation, sediment, and porewater from the *S. alterniflora* zone at all sites. At sites 1 and 3, we also collected samples for aboveground vegetation, sediment, and porewater from the *S. patens* zone of the marshes. Sediment cores from sites 2–5 were also sieved for roots and rhizomes in order to analyze BSi content in belowground vegetation. In all cases, triplicate measurements of each of the parameters were sampled. Sampling was paired, so that in all cases where we sampled aboveground vegetation, samples for sediment, belowground vegetation, and porewater were also collected. Sediment cores and porewater were sampled from within the 0.25 m2 plot where aboveground vegetation was collected. The same marshes were sampled during each season, but not the exact same 0.25 m2 plot within each marsh, as our sampling was destructive (e.g., removal of grass and sediment from marsh). All samples from each individual marsh were collected on a single day. The triplicate values from each individual marsh were averaged for each season (spring and summer). These average values for spring and summer were treated as distinct value for all analyses.

We harvested above ground vegetation from three randomly chosen 0.25 m2 plots per sampling event. Vegetation was washed thoroughly with deionized water and dried at 70◦C for 48 h before it was ground using a Wiley Mill. We collected sediment cores using PVC corers (30 cm long, 5 cm diameter). We divided our cores into four sections—the top 1, 1–10, 10–20, and 20–30 cm. In order to calculate bulk density (mass of material per unit volume), we dried each core section at 70◦C until it reached a constant mass over two consecutive days of weighing. Next, the cores were wet-sieved (0.5 mm sieve) to isolate roots and rhizomes, which were subsequently washed, dried, weighed, and analyzed for BSi concentrations. We collected triplicate porewater samples using porewater peepers, a passive sampling technique (Templer et al., 1998). We followed methods detailed in Carey and Fulweiler (2013), where 20-mL scintillation vials are pre-filled with deionized water and capped with a permeable membrane in order to allow the exchange of ions. Each polyvinyl chloride (PVC) peeper contained 10 vertically-stacked vials placed 3 cm apart. Peepers were deployed vertically in the sediment for 2–3 weeks, after which time the water was filtered using a 60-mL polypropylene syringe through a 0.45 micron nitrocellulose filter. The timing of peeper deployment always corresponded with collected of the other samples (e.g., sediment cores, aboveground vegetation). Porewater samples were stored in polyethylene bottles and kept in a cool dark place until they were analyzed for DSi. At sites 2–5, we also measured salinity and pH of all porewater samples.

We quantified sediment amorphous Si (ASi) content [which includes both BSi and pedogenic Si (Cornelis et al., 2010a)] and biomass BSi concentrations using the wet chemical alkaline extraction in 1% Na2CO3 solution (Demaster, 1981). Biomass was digested for 4 h. Sediment was digested for 5 h, with subsamples taken at hours 3 and 4 in order to calculate a mineral correction (Demaster, 1981; Conley and Schelske, 2001). We used a Seal AA3 flow injection analyzer and the molybdenum blue colorimetric method (Strickland and Parsons, 1968) to measure DSi concentrations. We routinely compared our standards to Hach external standards and they were always within 4% of the expected value.

In addition to our collected data from the five marshes in New England, we gathered all known published studies reporting aboveground plant tissue *Spartina* BSi concentrations and either productivity (biomass per unit area) and/or porewater DSi concentrations. In total, we located five studies that fit these characteristics (**Table 2**). The five studies presented data from salt marshes on three continents—Europe (i.e., the Netherlands, France), North America (i.e., New England and Southeast, USA), and Asia (i.e., China). We then identified general site characteristics of each site (e.g., bulk density and N availability) for comparison (**Table 1**).

In order to test the hypothesis that primary productivity or *insitu* Si availability was driving aboveground BSi concentrations in *Spartina*, we ran correlation analysis between BSi concentrations in aboveground material and the following variables: primary productivity [a proxy for plant age for perennial grasses, which reach maximum productivity in August and senesces in the fall (Wigand et al., 2004)], ASi concentrations in the sediment, and DSi concentrations in porewater. We also ran simple linear regression and used the least squares method to estimate model parameters, with *Spartina* BSi concentrations as the independent variable in all cases. In addition to examining all data together to see if broad trends in *Spartina* BSi behavior were apparent, we grouped our data by species and region to determine any differences related to taxonomy or location. We used the slopes of the regressions between *Spartina* BSi concentrations and porewater DSi concentrations to identify the mode of Si accumulation in these plants, with strong positive slopes indicating passive accumulation, negative slopes indicating active accumulation and flat slopes indicating rejective accumulation. Comparisons across sites were determined using a One-Way ANOVA and "multcompare" command in Matlab. All statistics were done using Matlab using α of 0.05 as the threshold for significance. All BSi concentrations reported as percent SiO2 by dry weight (dry wt.).

# **RESULTS**

# **Si CONTENT OF NEW ENGLAND** *S. ALTERNIFLORA*

A large range of aboveground BSi concentrations in *S. alterniflora* were found in the marshes, and concentrations typically increased throughout the growing season (**Table 3**). The minimum concentration observed was 0.24% by wt. (Site 5 in spring) and the maximum concentration measured was 1.04% by wt. (Site 2 in summer). These values align well with values reported in a metaanalysis by Hodson et al. (2005) and show that compared to other plant species in the order Poales, *Spartina* has relatively low Si concentrations.

Sediment ASi concentrations were almost always higher in the top 1 cm of the sediment compared to the depths of 1–30 cm (except Site 3 in spring) (Table S1). We found no significant differences in ASi concentrations seasonally or among sites, except for Site 5, which had ASi concentrations (0*.*35 ± 0*.*07%) an order of magnitude lower than all other sites in this study (Avg. 2*.*28 ± 0*.*47% by wt.) (**Table 3**, Table S1). Similar to the sediment ASi, the top one cm of the roots always had higher BSi content than the deeper depths (Table S2). Across all sites and both seasons, root BSi values ranged from a minimum of 0.18% observed at Site 5 in the summer to maximum value of 4.83% observed at Site 4 in summer. The average root BSi concentration across all sites and seasons was 1*.*03 ± 0*.*31%. Consistent with earlier observations in the same region (Carey and Fulweiler, 2013), the rhizomes always had significantly less BSi than the roots (Avg. 0*.*26 ± 0*.*03% by wt.) (Table S2). We found no seasonal or site related patterns to BSi concentrations in roots and rhizomes (Table S2).

Porewater DSi concentrations were higher during the summer compared to the spring and porewater values from Site 1 were typically higher than from the other marshes (**Table 4**, Table S3). Porewater concentrations ranged from 0μM (top 1 cm at Site 2 in spring) to 384μM (depth of 27 cm depth at Site 1 in summer) (Table S3). Across marshes, porewater pH was significantly (*p <* 0*.*01) different during both spring and summer seasons. For example, during the summer the pH at Sites 4 and 5 was significantly (*p <* 0*.*01) lower compared to Site 2.

#### **Si CONTENT OF NEW ENGLAND** *S. PATENS*

At two of the marshes in this study (Sites 1 and 3) we analyzed samples for Si accumulation in *S. patens*, or the high marsh platform zone of the marsh. Overall, Site 1 had higher Si accumulation than Site 3. For example, aboveground BSi concentrations of *S. patens* were roughly three times higher at Site 1 (**Table 3**), ranging from 0.58 to 1.25% by wt. (Site 2) and 0.21 to 0.43% (Site 3). Likewise, sediment ASi concentrations were also higher at Site 1 (Avg 2*.*16 ± 0*.*35% by wt.) than at Site 3 (Avg 1*.*31 ± 0*.*27% by wt.) (**Table 3**, Table S1). Similar to *S. alterniflora*, the ASi concentrations in the top one cm of sediment were always higher than the deeper layers of the core