Interactions between plant genome size, nutrients and herbivory by rabbits, molluscs and insects on a temperate grassland

Abstract

Angiosperm genome sizes (GS) vary ca 2400-fold. Recent research has shown that GS influences plant abundance, and plant competition. There are also tantalizing reports that herbivores may select plants as food dependent on their GS. To test the hypothesis that GS plays a role in shaping plant communities under herbivore pressure, we exploit a grassland experiment that has experimentally excluded herbivores and applied nutrient over 8 years. Using phylogenetically informed statistical models and path analyses, we show that under rabbit grazing, plant species with small GS generated the most biomass. By contrast, on mollusc and insect-grazed plots, it was the plant species with larger GS that increased in biomass. GS was also shown to influence plant community properties (e.g. competitive strategy, total biomass) although the impact varied between different herbivore guilds (i.e. rabbits versus invertebrates) and nutrient inputs. Overall, we demonstrate that GS plays a role in influencing plant–herbivore interactions, and suggest potential reasons for this response, which include the impact of GS on a plant's response to different herbivore guilds, and on a plant's nutrient quality. The inclusion of GS in ecological models has the potential to expand our understanding of plant productivity and community ecology under nutrient and herbivore stress.

1. Introduction

Terrestrial ecological communities are shaped by interactions between plants and herbivores, in which the availability of resources plays a central role (e.g. [1]). These interactions and their components are driven by a number of biotic and abiotic, top-down and bottom-up factors [2], one of which is nutrient availability, where a near-universal pattern following nutrient enrichment is an increase in plant biomass and a decrease in plant species diversity [3,4]. Nutrient availability also mediates the impact of herbivores on plant biomass and on community structure. For example, plants with access to nutrient resources are better able to tolerate herbivory [5]. In addition, grazing pressure on fertilized plant communities is associated with increased plant functional diversity [6]. Herbivores can counteract a decrease in plant species loss following nutrient enrichment by keeping fast-growing plants in check and promoting the growth of less competitive, but better-defended taxa [7,8]. Conversely, higher nutrient concentrations promote investment in plant growth rather than plant defences, increasing leaf palatability [9]. The production of enzymes and other proteins in growing plant tissues can also increase nitrogen (N) and phosphorus (P) concentrations, and such nutritious tissues are often favoured by herbivores, especially during the growing season (e.g. [10]).

Numerous studies have shown that certain plant functional traits can influence and mediate plant responses to stress caused by herbivory (e.g. [11,12]), and since plants form the basis of terrestrial food chains, any factor that can influence plant abundance and productivity has implications for all trophic levels. One factor that has been little explored in considering plant–herbivore interactions is plant genome size (GS; i.e. the amount of DNA in the unreplicated haploid nucleus—1C-value), which ranges ca 2400-fold between plant species [13]. Genome size can impact a wide diversity of plant traits, influencing how and where plants grow and interact across different ecosystems [14]. It may also impact how plant communities respond to grazing pressure, and plant–herbivore interactions. Certainly, there are tantalizing reports which suggest that GS can play a role in herbivore choice, although the responses are variable, e.g. some herbivorous insects favour polyploid over diploid cytotypes of the same species [15–18] or vice versa [19]; and cows may graze preferentially on a tetraploid versus diploid grass cytotype [20]. There are several reasons as to why plant GS might play such a role in plant–herbivore interactions. For example, plant species with very large genomes (e.g. 1C-values ≥ 25 pg (1 pg = 978 Mbp)) are slow growing, obligate perennials, perhaps due to longer DNA replication times [21]. Such plants are likely to recover poorly following herbivory compared with faster growing, more competitive species with smaller GSs. In addition, GS can influence leaf stoichiometry, as GS is positively correlated with leaf N concentrations [22], which could influence herbivore preference.

This paper explores the hypothesis that there are interactions between plant GS and herbivory that influence plant community composition and dynamics. We predict that plant communities under grazing pressure are primarily composed of species with smaller GS in contrast to communities where herbivores have been experimentally excluded, because tissue recovery from herbivore damage may be slower and costlier to produce in taxa with larger genomes and/or plants with larger genomes are nutritionally favoured by herbivores. We take advantage of an ongoing long-term ecological experiment which includes experimental manipulations of herbivory and nutrients (N and P). The experiment was established in 1992 at Nash's Field, Silwood Park, UK, on an acid mesotrophic grassland with intense herbivory by rabbits (Oryctolagus cuniculus) which are a keystone species. Our results reveal that GS clearly plays a key role in influencing interactions between herbivores, nutrients and plant biomass production. However, we also show that the direction of these interactions is dependent on the type of herbivore guild, with analyses at the plant community level revealing contrasting interactions and dynamics imposed by rabbit versus invertebrate (mollusc and insect) herbivory.

2. Methods

(a). Study site

The experimental study was started in 1992 on Nash's Field in Silwood Park, UK (National Grid reference 4 1/944691). Rabbits have been present at this site since their recovery from myxomatosis in the 1950s, and their grazing has prevented the establishment of woody species (e.g. Quercus species) and the succession from grassland to woodland. The experiment is set up in a split-plot, factorial design, comprising a total of eight herbivore exclusion treatments (± insect × ± mollusc × ± rabbits) (electronic supplementary material, figure SI.1). Each herbivore exclusion block (22 m²) is further divided in half with pH-controlled (limed at pH = 7, and unlimed at pH = 4.1) plots. In the first 3 years of the experiment, the limed plots also received herbicide treatments (± grasses, ± forbs). At the smallest plot level (4 m²) are the nutrient treatments, which comprise 12 combinations of: ±nitrogen (N) as ammonium nitrate (100 kg ha⁻¹), ±phosphate (P) (35 kg ha⁻¹), ±potassium (K) and ±magnesium (Mg), all of which are added once a year. Insects are controlled by a permethrin synthetic pyrethroid and dimethoate-40; molluscs (snails and slugs) by metaldehyde pellets; and rabbits by wire mesh fencing [23]. Insecticide and molluscicide are applied three times a year. Small mammals such as field voles (Microtus agrestis) and large mammals such as roe deer (Capreolus capreolus) are not excluded by rabbit fencing. The natural plant community is an intensely rabbit-grazed sward dominated by perennial C3 grasses, primarily Festuca rubra, but other common species include Agrostis capillaris, Holcus lanatus, Arrhenatherum elatius (Poaceae) and Jacobaea vulgaris (Asteraceae). We based our investigations on data from 190 limed, herbicide-free plots in the species-level analyses, and 556 limed plots in the plant community analyses.

We focus on limed plots rather than the acidic (pH 4.1) unlimed plots to avoid the confounding effects that would result from interactions between fertilization, in particular N, soil acidity and aluminium toxicity [24]. In addition, the analysis of limed plots enables comparisons with the only other analyses published on interactions between fertilizers and genome size in the generation of biomass [25,26], which are both also from temperate grassland field sites.

(b). Data collection

For analyses, we used biomass data collected in 1997 and 2000 from the 556 limed 4 m² subplots; we excluded 20 plots containing Pteridium aquilinum (an invasive fern) to focus on angiosperm plant communities. Where possible, species were sampled in 2015 to estimate their GS (1C-value) using standard flow cytometry methods ([27], see electronic supplementary material, table SI.1 for details). GS were estimated from one to eight individuals of 36 species collected from Nash's Field, with species reported to have several different ploidy levels being more extensively sampled. We used our own GS estimates when the coefficients of variation were less than 5% in the flow cytometry analysis, and obtained the remaining 1C-values from the prime values given in the Plant DNA C-values database [28] (electronic supplementary material, table SI.2). No intra-specific differences in ploidy level were found.

(c). Phylogenetic data

We pruned the DaPhnE phylogenetic tree [29], to include the 56 species present in the data with the R package ape [30]. We tested whether assumptions of a Brownian motion model were met (e.g. branch length is proportional to the amount of variation) with the caper package [31]. The most appropriate tree was one in which the branch lengths were transformed using the cladogram option in FigTree (http://tree.bio.ed.ac.uk/software/figtree/, version 1.4.3). We used this tree to account for non-independent evolutionary relationships among species in the statistical analyses (electronic supplementary material, figure SI.2). Phylogenetic signal in log-transformed 1C-values was significant (Pagel's λ = 0.761, p < 0.0001, Blomberg's K = 0.502, p = 0.001; estimated with phytools [32]).

(d). Data analysis

We first tested the effect of GS and experimental treatment on plant above-ground biomass at the species level. We then tested the effects of herbivory and macronutrient input on mean GS at the community (plot) level. Finally, we carried out path analyses to explore dynamics between plant community properties (sensu [33]): community-weighted mean (CWM) GS, CWM C-strategy (see below), total biomass, and phylogenetic diversity (PD), under the experimental treatments. Statistical analyses were carried out in R version 3.3.3 [34].

We refer to the C-strategy of a species as the competitor strategy in Grime's C—S (stress tolerant)—R (ruderal) plant strategies [35]. Each species is attributed to one, or more often, to a combination of these strategies (following [36]), based on how a species persists in its natural habitat, and where the sum of these values is equal to one (electronic supplementary material, table SI.2).

(i). Species-level analyses

We analysed the effect of GS, herbivore exclusion, and nutrient treatment on plant biomass at the species level using phylogenetic generalized linear mixed models with Bayesian estimation, by fitting Markov chain Monte Carlo generalized linear mixed models from the R package MCMCglmm [37]. Ideally, we would have analysed all the species in the model here, but this was not possible because many species were very rare, and in an experiment containing 556 plots, this gave a dataset with many absences (zeros). Statistically, comparisons of treatment effects on species biomass cannot be made on species with such few occurrences, preventing us from testing whether the treatment is having an effect versus whether species occurrence is random. Consequently, and after excluding plots where herbicides had been applied, (i) we limited the analysis to those species that occur on two or more herbivore treatments. This criterion still left the data too zero-inflated for analysis because so many species were so uncommon that rounding to the nearest tenth of a per cent equated to 0 (see electronic supplementary material, table SI.12). Therefore (ii) we limited the analysis to the most common species that had at least 1% of biomass on two or more plots. Together these two criteria did enable the model to converge, leaving for analysis a total of n = 12 species analysed over 190 plots. The results, while being restricted to the common species, generated data that support, and are fully congruent with, the community-level analyses in the forthcoming sections, where all species, their GS and biomass data are considered together in the same models.

We analysed species biomass in two-parts, similar to a hurdle model. Species presence/absence was first fitted in a logistic model, testing the probability of a species occurring on a plot, given the experimental treatments. The second part uses biomass data and fits only the non-zero biomass of the 12 species. The biomass data were log-transformed and analysed using MCMCglmm, assuming a Gaussian distribution. The exclusion of insects, molluscs, rabbits and the application of N, P and K fertilizer were scored as binary factors, with untreated plots coded as the reference levels. Genome size was centred on the median GS of the 12 species. Evolutionary non-independence was controlled for by specifying a correlation matrix estimated from the phylogeny. Random effects were specified as: block + block × fencing + species + phylogeny.

We first tested four-way interactions between GS, N, P and herbivore exclusion. Phosphate showed no significant interactions with herbivore exclusion, consequently, models were re-fitted without P in these interactions. Similarly, Mg had no effect on species occurrence and biomass and was removed to simplify the model. Fixed effects were thus specified as: (GS × insecticide × N) + (GS × molluscicide × N) + (GS × fencing × N) + (GS × N × P) + (molluscicide × insecticide × fencing) + K. We used priors where ν = 0.002 and variance = 1 [37] and ran the model with 2.5 million generations including a burn-in of 30 000 and a thinning interval of 500. We ran three chains for each model and assessed multiple chain convergence and trace and autocorrelation plots [38].

(ii). Community-level analyses

We investigated the effects of GS, herbivore exclusion and nutrient treatment at the community level. Each plot is representative of a plant community growing under various combinations of nutrient availability and herbivore guilds. For each plot (n = 556), CWM GS was estimated using the phylogenetic generalized least-squares method (PGLS). We fitted regressions with a Brownian motion correlation structure derived from the phylogeny [39], and maintained the same phylogenetic correlation structure across all plots. Species GS was log-10 transformed, and species per cent biomass was used for weighting: gls (log₁₀ GS ∼ 1, cor = corBrownian (phylogeny), weights = varFixed(approx. 1/species biomass)). We then back-transformed this mean, for use in the subsequent analyses and figures, to facilitate interpretation. To assess whether CWM GS is a function of herbivory and nutrient application, we fitted linear mixed effect (LME) models with the lme4 package [40] where each herbivore type (insect, mollusc, rabbit) and each nutrient (N, P, K, Mg), were scored as binary factors, and with random effects reflecting the split-plot design (plot/fencing/herbicide). Herbicide treatment was also included as a fixed effect, to account for its application in the early years of the experiment. Interactions between herbivore and nutrient treatments and the significance of each factor were tested using maximum-likelihood (ML) stepwise model reduction methods, and the final most reduced model re-fitted with restricted ML. Estimations of parameter significance (p-values) were obtained with the lmerTest package [41].

(iii). Confirmatory path analysis

To determine how herbivore guilds, nutrient availability and GS impact plant communities, we examined the effects of N, P, K and: (1) rabbit exclusion and (2) mollusc and insect exclusion on plant community structure using confirmatory path analysis. Specifically, we tested (i) whether GS influenced the abundance of plants with a C-strategy (sensu Grime [35]) at the community level (e.g. [25]); (ii) how the abundance of plants with a C-strategy impacts total biomass and PD and (iii) the role of herbivory and nutrients on these community properties. Data were partitioned into two datasets: (1) unfenced plots and fenced plots (rabbit exclosures) to test the effect of rabbit exclusion (n = 144 plots without insecticide or molluscicide treatment) and (2) ±insect and ±mollusc treatment (within rabbit exclosures only) (n = 281).

We examined four plant community properties: (i) CWM GS; (ii) CWM C-strategy; (iii) total above-ground community biomass (estimated from total dry sample weight) and (iv) PD. CWM C-strategy of each plot was calculated from each taxon's C-strategy (estimated with PGLS, as described above). PD corresponds to Faith's PD [42] and is the sum of the phylogenetic branch lengths of each community, estimated with the picante package [43].

We used directional separation (d-sep) path analysis methods [44] to assess 14 hypotheses about the effects of experimental treatment (herbivore exclusion, N and P plus K fertilizer) on the plant community properties (electronic supplementary material, figure SI.3, see also electronic supplementary material, table SI.3 for a more complete description of d-sep methods). The conditional independencies were fitted with LME models [45,46]. Random effects were specified as plot/fencing/herbicide in the rabbit exclusion analyses, and as plot/herbicide in the insect–mollusc exclusion analyses. Herbicide treatment was also accounted for as a fixed effect. The experiment was fitted as herbivore treatment × N × P + K + herbicide; where herbivore treatment = ±rabbits, or ±insects ×±molluscs. Continuous independent variables were standardized by two standard deviations [47] with the ‘rescale’ function from the arm package [48]. Because p-values are the main determinant in assessing acyclic diagrams, each equation was also systematically fitted with 10 unequal variance structures using the ‘varIdent’ function from the nlme package [39] to account for heteroscedasticity in the residuals (electronic supplementary material, table SI.3). We retained the p-value from the regression resulting in the lowest second-order AIC (AICc), which corrects for small sample sizes, implemented in MuMIn [49], but only if an analysis of variance showed that a regression fitted with the variance structure was significantly better (p < 0.05) than without. Interactions among community properties, and between the experiment and community properties, were not allowed. We present one directed acyclic diagram for each dataset, based on the CIC statistic and a preference for a more parsimonious diagram (electronic supplementary material, table SI.4), but this does not exclude alternative hypotheses that passed the goodness of fit tests. The conditional R²-values for LME equations were estimated with MuMIn and are measures of how much variance is explained by both fixed and random effects [49].

3. Results

Of the 56 species collected during the field sampling, 12 species generated at least 1% mean biomass in at least two herbivore treatment plots. Seven were grasses (Poaceae): Agrostis capillaris, Arrhenatherum elatius, Dactylis glomerata, Festuca rubra, Holcus lanatus, Holcus mollis and Phleum pratense subsp. bertolonii. The remaining five were Cirsium arvense, Jacobaea vulgaris (Asteraceae), Plantago lanceolata, Veronica chamaedrys, (Plantaginaceae) and Rumex acetosa (Polygonaceae). Overall, for the 56 species, GS ranged from 0.28 pg/1C in Juncus effusus to 11.06 pg/1C in Ranunculus repens (electronic supplementary material, table SI.2).

(a). Species occurrence and species biomass are a function of interactions between genome sizes, herbivory and nutrients

We investigated the impact of herbivores, nutrient treatment and species GS on the occurrence and biomass of the 12 species listed above. Herbivore exclusion and GS significantly impacted species occurrence. With insect exclusion, the probability of a species occurrence (inv.logit(B) = 0.813 (95% credible intervals (CI) = 0.397, 0.974, pMCMC = 0.0425)) increased by 0.472, or 47.2% in comparison to control plots with all herbivores present (electronic supplementary material, table SI.5A). Two significant interactions between GS and herbivores were apparent: (i) GS and rabbit exclusion—species with larger GS were more likely to be present on plots without rabbits. Each pg increase in GS was associated with an 8.0% increased likelihood of a species occurrence (inv.logit(B) = 0.573, CI = 0.530, 0.613, pMCMC < 0.0002), in comparison to control plots (inv.logit(B) = 0.49, electronic supplementary material, table SI.5A); (ii) GS and mollusc exclusion—removing molluscs increased the probability of a species with a smaller GS being present. Each pg decrease in GS was associated with a 6.6% increase in species occurrence (inv.logit(B) = 0.427, CI = 0.387, 0.465, pMCMC = 0.0020, electronic supplementary material, table SI.5A).

Species above-ground biomass was influenced by interactions between GS, herbivory and N (electronic supplementary material, table SI.5B). In control plots, the estimated effect of GS was a ca 62% increase in species biomass per pg increase in GS (exp(B) = 1.618, CI = 1.19, 2.36, pMCMC = 0.0057). The above-ground biomass of the 12 species was further impacted by three interactions between GS and experimental treatments. On plots with all herbivores present, the addition of N increased species biomass by more than twofold (exp(B) = 2.33, CI = 1.186, 2.358, pMCMC = 0.0105). However, this was dependent on GS, with most of the biomass increase coming from species with small GS—on + N plots, biomass decreased by ca 23% per pg increase in GS (exp(B) = 0.765, CI = 0.608, 0.974, pMCMC = 0.0243; electronic supplementary material, table SI.5B). The second interaction was an increase in biomass of species with larger GS on plots without rabbits—species biomass increased by 21% per pg increase in GS in comparison to control plots (exp(B) = 1.2099, CI = 1.047, 1.396, pMCMC = 0.0117)). This contrasted with the third interaction observed as the amount of biomass produced by species with larger GS on plots decreased with mollusc exclusion—a biomass decrease of ca 14% per pg increase in GS was observed when molluscs were excluded compared to control plots (exp(B) = 0.8604, CI = 0.736, 0.987, pMCMC = 0.0466)). Insect removal did not significantly affect the growth of the 12 species.

(b). Herbivory and nitrogen influence community-weighted mean genome sizes

For community-level analyses, CWM GS were estimated for all 556 plots using biomass values for the 56 species; these ranged from 1.36 pg/1C (on a plot with + N and –rabbits and –molluscs (i.e. insects only) to 8.61 pg/1C (on a plot with + N and –rabbits). For control plots (all herbivores present, no nutrient input), the CWM GS was estimated at 5.76 pg/1C (95% confidence interval (CI) 5.02, 6.50) (electronic supplementary material, table SI.7).

The most parsimonious LME model (obtained with stepwise reduction, electronic supplementary material, table SI.6) revealed that: (i) on unfertilized plots, the biomass from plants with larger GS increased without rabbits—CWM GS of plots increased by 1.10 pg/1C when rabbits were excluded compared to control plots (figure 1, CI = 0.26, 1.95, p = 0.0538, electronic supplementary material, table SI.7). This effect became significant with the addition of N, with CWM GS of plots increasing by 0.33 pg/1C with rabbit exclusion, compared to control plots (CI = 0.46, 1.73, p = 0.0008). (ii) By contrast, the biomass from plants with smaller GS increased on plots with both mollusc and insect exclusion and N input. The CWM GS of plots decreased by 2.73 pg/1C on + N plots with rabbit grazing only (B = 1.433, CI = 0.52, 2.35, p = 0.0024). (iii) Nitrogen fertilizer decreased CWM GS of plots with all herbivores present by 1.87 pg/1C (CI = −2.42, −1.32, p < 0.0001) and (iv) P fertilizer decreased CWM GS by 0.31 pg/1C (CI = −0.54, −0.08, p = 0.0078). (v) CWM GS of plots with only insect grazing (rabbit and mollusc exclusion) and N fertilization decreased by 1.54 pg/1C compared to control plots (figure 1, CI = −2.10, −0.28, p = 0.0114, electronic supplementary material, tables SI.7).

Figure 1.

Community-weighted mean genome size (GS; 1C-value, pg) of each plot (n = 556) under eight herbivore exclusion treatments and ±nitrogen (N) input. Herbivore treatments, from right to left are: (1) control (all herbivores present); (2) – insects; (3) – molluscs; (4) – rabbits; (5) rabbits only (– insects, – molluscs); (6) molluscs only (– insects, – rabbits); (7) insects only (– molluscs, – rabbits); (8) no herbivores. Boxes show median of community-weighted mean GS, first and third quartiles, and minimum and maximum values (summary statistics in electronic supplementary material, table SI.8). (Online version in colour.)

We also estimated CWM GS (i) without including a phylogenetic correlation structure; (ii) with GS untransformed; (iii) with the lambda parameter optimized; and showed that these were similar to CWM GS used above and that the key results presented here remain unchanged (electronic supplementary material, figure SI.4 and table SI.9).

(c). Contrasting and similar effects of rabbit and invertebrate herbivory on plant community properties

Using confirmatory path analysis, we investigated how the following four plant community properties were influenced by each other and by herbivory and nutrient treatments: (i) CWM GS, (ii) CWM C-strategy (i.e. species competiveness), (iii) total above-ground community biomass and (iv) PD (figure 2a,b; electronic supplementary material, figure SI.5 and tables SI.10 (rabbit exclusion) and SI.11 (insect and mollusc exclusion) for complete regression tables of figure 2a and b respectively).

Figure 2.

Path analysis examining effects of (a) rabbit exclusion and (b) mollusc and insect exclusion together with nutrient input (N, P, K) on four plant community properties: (i) community-weighted mean (CWM) genome size (GS), (ii) CWM competitive (C)-strategy, (iii) total biomass and (iv) phylogenetic diversity. Values in ovals show the effect size of one variable, or interaction, on the other. The conditional R² and the intercept are given for each community property. Arrow widths are proportional to standardized coefficients. P-values: ***<0.0001, **<0.001, *<0.05 (see electronic supplementary material, tables SI.10 and SI.11 for details in (a) and (b), respectively). (a) For rabbits, a three-way interaction (± rabbits × N × P) did not contribute significantly towards any community property, thus only two-way interactions were included between ± rabbits, N and P. (b) For molluscs and insects, CWM GS could not be unlinked from total biomass in a conditional independency test, however its partial regression coefficient is below the alpha level (0.050 at p = 0.0709). (Online version in colour.)

In testing the impact of rabbits, the four community properties were influenced by various factors. The interaction between N input and rabbit exclusion increased CWM GS by 0.99 pg (CI = 0.44, 2.24, p = 0.0047; figure 2a; electronic supplementary material, table SI.10). CWM C-strategy increased with N input (B = 0.089, CI = 0.04, 0.15, p = 0.0016), and with CWM GS (B = 0.025, CI = 0.02, 0.04, p < 0.0001), particularly on plots without rabbits (electronic supplementary material, figure SI.6a, b). Total biomass increased with rabbit exclusion and N input (B = 19.97, CI = 10.85, 29.08, p = 0.0001). The single largest standardized effect is the reduction in PD with rabbit exclusion (B = −15.48, CI = −22.16, −9.54, p = 0.0003).

By contrast, removal of molluscs and insects did not reduce PD. Instead, N input, and to a lesser extent, K input, were the main drivers of this (B = −12.11, CI = −18.54, –5.68, p = 0.0004; and B = −3.58, CI = −6.02, −1.15, p = 0.0052, respectively; figure 2b; electronic supplementary material, table SI.11).

The main driver influencing occurrence of species with a competitive life strategy (i.e. CWM C-strategy) was a four-way interaction between insect and mollusc exclusion and +N and +P input, increasing CWM C-strategy by 0.23 units (CI = −0.46, −0.11, p = 0.002). Similar to ±rabbit plots, CWM C-strategy also increased with CWM GS (B = 0.025, CI = 0.02, 0.03, p < 0.0002; electronic supplementary material, figure SI.6c–f). Total biomass increased significantly with both +N and mollusc (but not insect) removal by a mean of 28.05 g (in dry weight) (p = 0.0073). In addition, increased biomass was associated with increased CWM C-strategy (B = 65.65, CI = 43.63, 85.97, p < 0.0001). Finally, in contrast to rabbit exclusion, a four-way interaction between +N, +P, insect and mollusc exclusion led to a decrease of 2.39 pg in CWM GS (p = 0.0464). Total biomass and PD were not significantly associated with each other in both path analyses.

(d). Changes in species composition with herbivore exclusion and N fertilizer

Species that increased with rabbit exclusion, include the larger genomed Arrhenatherum elatius (8.61 pg/1C), and to a lesser extent H. mollis (4.1 pg/1C), and D. glomerata (4.4 pg/1C). The response of F. rubra (7.31 pg/1C) was dependent on N, decreasing on −N plots, and increasing on +N plots. The removal of molluscs led to an increase in the smaller genomed H. lanatus (1.7 pg/1C), followed by P. pratense subsp. bertolonii (1.99 pg/1C) and H. mollis (4.1 pg/1C), at the expense of the larger genomed F. rubra and A. elatius. By contrast, species that increased most with just insect removal had a range of GS and included A. elatius (on –N plots), H. mollis (on +N plots), J. vulgaris (2.25 pg/1C), C. arvense (1.42 pg/1C) and H. lanatus; while A. capillaris (3.6 pg/1C) and F. rubra decreased.

Within fenced plots, species that increased with removal of molluscs and insects include H. mollis, H. lanatus, D. glomerata, Achillea millefolium (7.98 pg/1C)), P. pratense subsp. bertolonii and V. chamaedrys (2.16 pg/1C). Holcus mollis increased consistently on +N plots, whereas F. rubra, A. capillaris, C. arvense (1.42 pg/1C), decreased on −N plots (see also electronic supplementary material, figure SI.7 for biplots representing species abundances for the experimental treatments, and electronic supplementary material, table SI.12 for percentage change in species biomass).

4. Discussion

The experiment at Nash's Field involves the input of nutrients and the exclusion of grazing by molluscs, insects and rabbits, allowing us to detect significant interactions between GS, herbivory and N fertilization. We show that these interactions impact plant community structure, plant biomass production and species diversity. Previous work showed that plant community structure was influenced by interactions between plant GS and nutrients directly, and that species with larger GS only contributed significant biomass to plant communities when nutrients were not limiting [25,26]. We did not observe this effect here, perhaps because: (i) the experiment is still young (data were collected only 6–8 years after the start of the experiment), compared with the Park Grass (greater than 150 years) and Rengen Grassland (greater than 70 years) Experiments, and thus plant communities may still be adapting to the experimental treatments and are in a transient state [45]; (ii) since the 1950s intensive rabbit grazing is known to have been prevalent in the area used to establish Nash's Field Experiment in 1992 and this would have influenced the species that colonized the plots towards those that are grazing-tolerant and/or grazing-resistant [46,50].

(a). Impact of rabbit herbivory

We observed that the plant species that generated most biomass on rabbit-grazed plots had smaller GS than those on ungrazed plots, especially when N fertilizer was added. This could be because:

• (i)Rabbits preferentially eat plant taxa with large GS. Plant species that increase in biomass when herbivores are excluded are generally species that are preferentially grazed by herbivores [51]. Rabbits are known to favour high-nutrient plants [52]. N input qualitatively alters plant nutrient content [53] and plants growing under higher N may be more attractive to consumers and increase herbivore numbers [10,54]. Potentially rabbits may prefer species with larger GS because at the cellular level, they have higher N and P content (both DNA and RNA are rich in these macronutrients) compared to species with smaller GS [53,55]. In addition, plants with larger cells may be more palatable as cell size tends to increase with GS, and larger and fewer cells per leaf would decrease the amount of cell wall, potentially rendering the plant more succulent.
• (ii)Plant taxa with smaller GS are better able to recover from the rabbit grazing pressure. Rapid growth might be best achieved in plants with small GS which have faster cell cycle times, shorter durations of DNA synthesis, and hence shorter minimum generation times (reviewed in [14]), and may be less constrained by nutrient availability for growth and repair [56]. Indeed, tolerance to grazing may be a key survival trait on fertilized plots, where rabbit abundance can be four times greater than on unfertilized plots [57]. Rabbits generate sustained stresses that impact grassland composition and dynamics, such as the selection of plants with rapid growth rates [58]. Rapid growth rate is fundamental to tolerance of herbivory, allowing for regrowth of damaged tissue. Such regrowth is achieved via rapid resource allocation, increased photosynthetic rates and increased nutrient uptake [59]. Trade-offs may, therefore, exist between the biochemical costs associated with building and maintaining a large genome and tolerance to herbivore damage.
• (iii)Plant taxa with smaller GS may be less constrained to allocate resources to the production of secondary metabolites for defence. Investment in defence has been shown to lead to decreased rates of photosynthesis, and the diversion of elemental resources such as C and N towards the production of defence compounds at the expense of growth [60]. Defence is also costly in both water and nutrient resources [61], leads to the remobilization of elements to roots [62], and would compete with the N and P costs of building and maintaining a large genome, especially in nutrient-deficient environments.

In addition to the direct effects of preferential grazing, rabbits increase habitat and resource heterogeneity by trampling, burrowing, decreasing ground cover and deposition of droppings. These effects may enable functionally more diverse species to colonize the community [7]. We observed that the presence of rabbits leads to a significant increase in PD, which is also associated with a decrease in plant species with a competitive life strategy and a decrease in total biomass (figure 2a). Rabbit grazing on the fast-growing, dominant plants keeps these in check, allowing the establishment of subordinate species. This effect has also been reported across various types of grasslands with mammalian herbivores, but depends on plant productivity, generally being positive in grasslands with high productivity, but decreasing plant diversity when productivity is low [63,64]. Experiments are now needed to determine whether plants with larger GSs, are indeed of higher nutrient quality, altered N and P contents and different recovery periods following damage by herbivory.

(b). Impact of invertebrate herbivory

In contrast to rabbit grazing, mollusc and insect grazing leads to communities composed of plants with larger GS. In the absence of fertilizer, mollusc and insect herbivory appear to mitigate the effects of rabbit grazing, as plant communities on plots with no herbivores have similar CWM GS as the control plots where all herbivores are present (figure 1). Numerous studies have shown that molluscs have species-specific food preferences, which relate to different food qualities and a plant's palatability, the latter being influenced by, for example, the presence of plant anti-herbivory chemicals, silica content and a plant's pubescence which can deter feeding [65]. Molluscs are also reported to have a preference for seedlings, however, in a temperate grassland seedling recruitment is low, where plant regeneration is mostly via vegetative regrowth [23]. Potentially, the invertebrates at Nash's Field are specialists on plants or plant organs that are not favoured by rabbits. Nutrient acquisition strategies vary between and within these herbivore guilds, and the scale and impact of different insect herbivores (predominantly grasshoppers) on grassland plant community biomass has been shown to be linked closely with, for example, mandibular trait diversity [66]. It is possible that insects as a guild feed on a larger range of species and tissues than molluscs as a guild, making the effects of insects on community plant GS more difficult to detect.

While insect and mollusc herbivory decreased total community biomass, the effects of insects and molluscs on community composition were negligible when fertilizers were added, perhaps because fertilizers can obscure the effects of nutrient recycling by grazers, although interactions are complex [1]. The application of N and K led to a reduction in PD, for which the presence of insects and molluscs did not compensate. This is opposite to the significant effect of rabbits; their larger size and activities (as noted above) may result in more pronounced effects on plant diversity [63]. Previous findings on the unlimed plots at Nash's Field, included a decrease in species diversity with insect removal, and, to some extent, mollusc removal [23]. The effects of insects and molluscs may take longer to occur on limed plots, as these do not have the additional stress of low pH conditions.

(c). Genome size and ecological models

Investigations of plant traits have uncovered global trends that help to predict plant responses to abiotic and biotic factors [67–69]. Here we propose that GS should also be considered as an important plant functional trait which influences plant community structure through bottom-up (nutrients) [70] and top-down (herbivory) interactions with GS. We show here that larger CWM GS is linked with higher CWM C-strategy, which is, in turn, linked with increased community biomass. Higher species competitiveness is also associated with decreased species diversity in the absence of rabbit grazing. Through plant consumption, herbivores also influence plant community composition, including the abundance of plants with a competitive life strategy, which can impact nutrient cycling by altering litter quality [1]. The effect of GS on plant tolerance to herbivory, and possible influence on herbivore preference may also have a wider ecological impact, influencing processes such as N cycling, and hence add a new dimension to improve the performance of ecological models.

Data accessibility

All data are available in the Dryad Digital Repository at: https://doi.org/10.5061/dryad.kd6k37v [71].

Authors' contributions

M.S.G. and D.K. carried out fieldwork and GS estimation. M.S.G. carried out the data analyses. M.J.C. established the experimental plots, assisted with species identification and provided biomass data. R.A.N. provided statistical advice and M.S.G., M.T., A.R.L. and I.J.L. conceived the fieldwork. M.S.G., I.J.L. and A.R.L. devised the analyses and wrote the manuscript. All authors read, revised and approved the final manuscript.

Competing interests

We have no competing interests.

Funding

This study was supported by funding from the Research Council of Norway (196468/v40) and the Natural Environment Research Council (NERC) (NE/J012106/1).