Plant production and community structure in a mesic semi-natural grassland: Moderate soil texture variation has a much stronger influence than experimentally increased atmospheric nitrogen deposition

doi:10.21203/rs.3.rs-3863615/v1

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Plant production and community structure in a mesic semi-natural grassland: Moderate soil texture variation has a much stronger influence than experimentally increased atmospheric nitrogen deposition

https://doi.org/10.21203/rs.3.rs-3863615/v1

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Aims

Atmospheric nitrogen (N) deposition derived from agricultural intensification and fossil fuel burning can significantly impact plant growth, species diversity, and nutrient cycling. Semi-natural grasslands are of particular concern because their generally low intensity agricultural management suggests they may be very sensitive to enhanced atmospheric N deposition inputs, although previous experimental research indicates highly variable site-specific responses. Mediating factors such as soil texture that influence actual availability of soil water and nutrients to plants have generally not been investigated.

Methods

We report the impacts of 16 years of experimental N addition (simulating 2050 atmospheric N input rates) to a hayfield of varying loamy soil texture (clay-loam – sandy-loam) on plant community structure and above-and belowground biomass.

Results

Chronic low-level N addition treatment had no significant effects, while species richness, diversity, and aboveground growth were best explained by variation in soil water-filled pore space and were substantially larger on those plots with clay-rich soils.

Conclusions

The consistent lack of responses to the low-level N additions and the lack of a growth response to a separate single year’s factorial high-level N and phosphorus addition experiment, indicate that future atmospheric N deposition increases are unlikely to have major impacts on hay production or species composition in mesic semi-natural grasslands. By contrast, the strong interconnected influences of soil clay content and water availability in our results suggest that texture variation – even within loamy soils - will be a primary determinant of the impacts of anticipated future summer warming and reduced rainfall on hayfield vegetation.

water availability

plant community composition

root biomass

productivity

atmospheric nitrogen deposition

soil texture

Grasslands cover between 25–40% of the Earth’s surface (LeCain et al. 2002; McSherry and Ritchie 2013; Soussana et al. 2010; Wang and Fang 2009), and they are characterized as highly productive ecosystems(Carrillo et al. 2014) with high levels of species diversity. In particular, semi-natural grasslands are global biodiversity hotspots(Neff et al. 2020) because of their unique communities of plant species, pollinating insects, and other wildlife that rely on key native grass species for nests, food, and shelter (Öster et al. 2007; Reitalu et al. 2010). Plant species composition in grasslands is also important because it influences productivity, resistance to invasion, and overall ecosystem health (Zavaleta and Hulvey 2007). Semi-natural grasslands tend to have low innate soil nutrient fertility, and therefore are ‘marginal’ for agriculture. Furthermore, these systems have a history of infrequent or no fertilizer nutrient inputs, and small but regular nutrient removal due to annual pasture grazing and/or hay harvesting. Accordingly, from a nutrient perspective, these small inputs and outputs suggest that semi-natural grassland plant community structure and productivity could be highly susceptible to cumulative nitrogen (N) additions associated with increased atmospheric N deposition (Bobbink et al. 2010).

Atmospheric N deposition refers to the inputs of reactive N from the atmosphere to the biosphere that are caused by heavy fertilizer additions and other intensive agricultural practices, as well as fossil fuel burning (Chapin et al. 2012). These anthropogenic activities now dominate the global N cycle and make atmospheric N deposition one of the most widespread drivers of global change (Galloway et al. 2008; Humbert et al. 2016). Increased N deposition rates over the past century have already caused ecologically significant enrichment of N across terrestrial and aquatic environments (Meng et al. 2012), and are jeopardizing both the structure and functioning of these ecosystems in many ways including habitat degradation(Bobbink et al. 1998; Sala et al. 2000) and facilitation of exotic species invasions (Chen et al. 2019). Therefore, understanding ecosystem responses to prolonged and increasing aerial N inputs is critical. Furthermore, the continued intensification of N-based anthropogenic activities into the future is expected to severely threaten not just the sustainability and health of terrestrial ecosystems (Bobbink et al. 1998, 2010; Sala et al. 2000), but also their resilience to the changing climate.

Our review of previous research on current and anticipated impacts of N deposition on natural and semi-natural grasslands indicates the following conclusions. First, grassland environments experiencing increased atmospheric N deposition generally display clear increases in productivity if N is the primary growth-limiting nutrient (Phoenix et al. 2012). Second, N deposition even at low levels, reduces species richness and diversity(Bai et al. 2010; Clark and Tilman 2008; Stevens et al. 2004, 2010; Wilcots et al. 2021) and drives individual species loss through competitive exclusion (Bobbink et al. 2010; Suding et al. 2005). Third, although, many individual fertilization studies have demonstrated that N is generally the primary nutrient limiting grassland plant productivity (e.g., Faust et al. 2012; Janssens et al. 1998), at a more broad global scale, productivity of ~¾ of all 42 experimentally tested grasslands across 5 continents were primarily limited by some combination of N, phosphorus (P), potassium (K), and micronutrients (Fay et al. 2015). In particular, synergistic co-limitation by N and P was common, contradicting the widely held view that N availability is generally the single most important plant growth-limiting factor in terrestrial ecosystems (Fay et al. 2015).

Responses of plant communities to elevated N deposition may be strongly mediated not just by other nutrients, but also by other limiting resources, as well as abiotic factors controlling the availability of those resources. For example, soil texture is a fundamental control over water and nutrient availability to plants (Baer et al. 2010; Bosatta and Ågren 1997; Brady and Weil 2008; Chapin et al. 2012). Consequently, differences in soil water holding capacity and matric potential among distinct soil textures are critical determinants of plant species composition and relative abundances because some plant species are typically better adapted to certain hydrological conditions than others (Chapin et al. 2012; Xia et al. 2010). The previous research described above has assessed individual plant community responses to realistic atmospheric N deposition rates (e.g., Bowman et al. 2012; Midolo et al. 2019; Prager et al. 2017; Wilcots et al. 2021), as well as multi-nutrient additions (e.g., Fay et al. 2015; Huang et al. 2018; Wilcots et al. 2021) but almost no studies have addressed the potentially mediating influence of soil texture. Furthermore, almost all investigations to date have focussed solely on aboveground responses (e.g., Clark and Tilman 2008; Zheng et al. 2019), with very few specifically incorporating belowground traits (Bai et al. 2015; Huang et al. 2018),even though shoot responses are indirect and ultimately driven by the direct effects of atmospheric N deposition on soil N availability belowground.

This study focusses on the long-term (16 year) impacts of simulated enhanced atmospheric N deposition on above- and belowground productivity, species diversity, and nutrient availability in a temperate southeastern Ontario semi-natural hayfield containing discrete sections of clay-loam and sandy-loam soil texture. To the best of our knowledge, no previous study has specifically investigated the potential influence of variability in soil texture in mediating potential effects of experimentally enhanced atmospheric N addition on soil N availability to plants (i.e., the potential interaction between enhanced N inputs and soil texture). Additionally, on the basis of earlier preliminary research at our study site indicating no effect of chronic low-level N addition on plant community production after nine years (Constant and Grogan, Unpublished results), we directly tested our a priori assumption that plant growth was in fact primarily limited by availability of N (and/or P) using a separate, single season, factorial high-level N and P addition experiment on both soil texture categories. We tested the following hypotheses:

Plant community productivity above- and belowground are each significantly affected by both chronic low-level N addition and field variation in soil texture, with the latter factor having the strongest influence.

Semi-natural grassland plant community species richness is significantly reduced by chronic low-level N addition, especially on the relatively species-rich clay-loam compared to sandy-loam soils.

Plant species diversity (i.e., richness combined with evenness) is determined by the interaction between chronic low-level N addition and soil texture, with the latter factor having the strongest influence.

Plant community aboveground production in our semi-natural grassland is primarily limited by soil N and/or P availability, but the responses are strongly mediated by soil texture.

Stoke’s Field Hay Grassland Site Characteristics and History

This research was conducted at the Stoke’s Field semi-natural grassland agroecosystem (44°30'48.4"N, 76°23'47.2"W) on the Canadian Shield in southeastern Ontario, Canada (Fig. 1). The region experiences a ‘Warm Summer Continental Climate’ (Koppen classification Dfb), with hot summers (mean 20.1⁰ C) and cold winters (mean − 5⁰C), and the mean annual precipitation of ~ 950 mm yr^− 1 which is evenly distributed across the seasons (Environment Canada 2021). Eastern Ontario is dominated by soils of low fertility that vary greatly in texture as a result of dynamic and heterogeneous post-glacial sediment deposition that occurred at the end of the Holocene (Cumming and Al-Aasm 1999). Stoke’s hayfield contains soils that are classified according to the USDA system as loams of varying texture (Constant and Grogan, Unpublished results, Table S1). The flat northern half of the field is on a ‘sandy-loam’ (8% clay, 22% silt, 70% sand), while the gently sloping southern half is on a ‘clay-loam’ (29% clay, 43% silt, 28% sand). Stoke’s Field has been cut for hay once per year since its acquisition by the Queen’s University Biological Station in 1976. It was probably under low intensity agricultural management (e.g., hay and light cropping/pasture) over the previous century and was likely sown with grass and forb species such as Timothy Grass (Phleum pratense), Tall Fescue (Festuca arundinaceae), and Red Clover (Trifolium pratense) (Phelan, personal communication).

Atmospheric Nitrogen Deposition Experimental Setup

The simulated chronic low-level enhanced atmospheric N deposition experiment was set up in 2005 and consists of 12 interspersed N addition and control plots (5 m x 4 m), distributed in two rows of six from north to south, across the two soil texture categories (Fig. S1, Table S1). This experimental layout was chosen to address possible impacts of different forest shading through the day associated with the tall banks of trees on the east and west sides of the field.

Enhanced atmospheric N deposition was simulated by adding 25 kg N ha^− 1 yr^− 1, which is 2.5 times the current deposition rate in southeastern Ontario and represents estimated rates for the year 2050 (Galloway et al. 2008). From 2005 to 2008, granular applications of 8.3 kg N ha^− 1 yr^− 1 were added three different times per year (Fig. S2): late fall, early spring, early summer. After 2008, the full addition of 25 kg N ha^− 1 yr^− 1 was applied at one time in late-May/early-June. Initially, 142.84 g of 35-0-0 (N-P-K) ammonium nitrate fertilizer (FS brand from Growmark Inc. Bloomington IL.) was added to each 20 m² experimental plot, and from 2017 onwards, the fertilizer-type was switched to urea 0-45-0 (CO(NH₂)₂) (FS brand from Growmark Inc. Bloomington, IL.) which was added at 108 g per plot.

Above- and Belowground Plant Collection and Mass Analysis

Total aboveground biomass and species composition were determined by harvesting a 1 m² quadrat within each plot (Fig. S3) between August 10th to 20th, 2020 in a structured random order. Each quadrat was divided into four equal sections and all aboveground plant material in one of those sections (subplot A) was clipped where the shoots met the ground and then sorted by species The remaining three sections of each 1 m² plot were clipped at the ground surface and collected without sorting to species. Only vegetation that was rooted within the plot was harvested and standing dead plant matter was not included. All plant samples were placed in a fan-assisted drying oven at 60⁰ C for ~ 8 days, then weighed.

After the aboveground harvest in each plot, a soil section (~ 0.3 m x ~ 0.3 m area to ~ 0.1 m depth) was removed from subplot A using a shovel (Protocol S1) to measure root biomass and the aboveground to belowground biomass ratio. Length, width, and height of each core were recorded at three different locations on each dimension to calculate the volume of the soil section removed. The cores were then transported back to the lab and frozen.

Belowground Biomass Sample Processing

See Protocol S1 in Supplementary Materials for full details. Briefly, the soil sections were prepared for root separation and sorting by thawing them completely and then soaking them in water for 8–24 hrs to facilitate separation of the soil from the roots. The soil sections were then washed and sieved three times to remove all soil and then sorted into rhizomes and fine root categories based on size, texture, and colour. All residual material (e.g., aboveground shoots, dead biomass, etc.) was discarded. Rhizome and fine roots were dried at 60⁰ C for > 3 days before weighing. Belowground biomass (g of root dry weight per cm³) and its components (fine roots, rhizomes) were transformed to grams of root dry weight per m² to allow for comparison with total aboveground biomass.

Ion Exchange Membrane Measurement of Soil Nitrogen and Phosphorus Availability to Roots

Cation exchange membranes (CEM) and anion exchange membranes (AEM) were used to measure ammonium, nitrate, and phosphate fluxes within the plots (SUEZ Water Technologies & Solutions, USA) on the 3rd, 10th, and 17th of July 2020. CEMs were charged in 0.5 M HCl solution while AEMs were charged in a 0.5 M NaHCO₃ solution. For the detailed Ion Exchange Membrane (IEM) protocol see Gu and Grogan (2020). On the edge of each plot, two vertical slits ~ 2 cm apart were cut into the soil with a serrated knife and a pair of IEM squares (25 cm² each) were inserted vertically from 2–7 cm underneath the soil surface at each incubation location (Fig. S3). The slits were then closed by pressing the soil surface to ensured good soil membrane contact. After a seven-day incubation, the IEMs were removed and rinsed with deionized water in the field before transport to the lab for fridge storage. Each IEM was eluted in 20 mL of 2 M NaCl / 0.1 M HCl solution following the protocol ofGu and Grogan (2020) and were then analysed for ammonium, nitrate, and phosphate concentrations using standard colorimetric methods. Tests of blank membranes (i.e., membranes that were not incubated in soil) showed contamination levels below detection limits.

Abiotic Measurements

Soil moisture (Volumetric Water Content %) was measured using a handheld soil moisture meter (HydroSense II, Campbell Scientific) with the 12 cm long probe inserted at a 45- degree angle to measure moisture content over the ~ 0–5 cm depth interval at three random locations in undisturbed areas of each plot. Likewise, soil temperatures were measured at 2 cm, 5 cm, and 10 cm depth at three random locations in each plot. Soil moisture and temperature values within in each plot were averaged to yield one datum per plot at respective depth intervals, and measurements were made on five separate dates between mid-June and early August and averaged to calculate mean growing season soil moisture and soil temperature.

Soil Texture Calculation and Classification

All particle size analysis data were determined using the buoyancy method (Gee and Bauder 1979) and classified using the USDA classification system. Most of these analyses were done previously (Constant and Grogan, Unpublished results) apart for plot C1 and five test re-analyses of plots from the earlier dataset, which were collected in 2021.

Soil Texture and Soil Moisture Interaction (Water Filled Pore Space)

Prior to beginning statistical model analysis, we determined that soil texture and soil moisture were moderately correlated (Overall Pearson’s Correlation r = 0.49, P > 0.0001), suggesting that an interaction between soil moisture and clay concentration may be an important determinant of variation in vegetation responses across the field. To investigate the interconnected influences of soil texture and moisture on both above- and belowground biomass, species diversity, and nutrient fluxes, we calculated percentage water-filled pore space (WFPS – the proportion of total soil porosity containing water) because it should integrate the effects of both factors across a range of soil textures and soil moisture conditions (Scott et al. 1996). We calculated WFPS (%) for each plot using mean volumetric soil water content, soil volume, and bulk density for each plot.

WFPS = VWC / (1-BD / PD)

Where:

WFPS = Water-Filled Pore Space (%)

VWC = Soil Volumetric Water Content (%)

BD = Soil Bulk Density (g/cm³)

PD = Particle Density (2.65 g/cm³)

Nitrogen And Phosphorus Growth Co-Limitation Study

To test for direct nutrient-limitation of plant community growth, we set up a separate, single year, high-level N and P factorial addition experiment (Fig. S1) with the various fertilizer treatment additions applied in a structured random order. Forty-eight 2 x 2 m plots (n = 6) were located in six rows between the chronic low-level N addition plots in both the clay-loam and sandy-loam sections of the field on May 20th, 2020. N (in the form of urea as detailed above) was added at a concentration four times higher than for the low-level N addition treatment (i.e., at a rate of 100 kg N ha^− 1, which is equivalent to 86.9 g urea fertilizer per 4 m² plot), while P (as “available phosphoric acid P₂O₅” 45%, FS Fertilizer) was added at a concentration of 50 kg P ha^− 1 which is equivalent to 103.6 g P₂O₅ -based fertilizer per 4 m² plot. Aboveground biomass of the entire plant community was sampled from a 1 m² quadrat in the center of each 4 m² plot from August 21st to 31st, 2020, and processed as described above.

Statistical Analyses

Effects of the chronic low-level N addition treatment, WFPS, their interaction, and soil temperature were tested on each of the following response variables: plant community biomass components, species richness, Shannon Diversity Indices, and the soil nutrient fluxes (Tables 1 and S2). The data for total aboveground biomass and Shannon Diversity Index met all assumptions to analyse using linear models, while total belowground biomass, rhizome biomass, fine root biomass, and soil ammonium fluxes were analyzed using generalized linear models after applying an inverse gaussian distribution. Soil nitrate and phosphate fluxes were also analyzed using generalized linear models but after applying a gamma transformation and an inverse-link function (g(µ) = 1/µ), while species diversity was analyzed after applying a Poisson distribution function (Table S2). In terms of potential correlation among explanatory variables, we compared the fit of soil moisture, soil clay concentration, and WFPS in all models since these three properties are inter-related, and found the latter was the best predictor. Therefore, we report the WFPS-only analyses in the Results. We evaluated each set of competing models using Akaike’s information criterion (AIC) calculated with the ‘AICc’ function in the MUMIN package (v.1.46.0, Burnham and Anderson 2004). When multiple competing models occurred with AICc delta ≤ 2, the best-fit model was selected though careful evaluation of Akaike weights, variable importance, and likelihood ratios. P-values and F-statistics for each model were obtained using the ‘Anova’ function in the car package (v.3.0–13, Fox and Weisberg 2019). After each analysis, we tested model validity by visually inspecting the distributions of residuals and of the residuals plotted against predicted values and found no obvious outliers or deviations from the assumptions of normality or homogeneity. All statistical analyses were performed using R version 4.0.5 (RStudio Team 2021), and all statistically significant (p < 0.05) main effects and interactions are reported in the Results.

Overall differences in all response variables between the above- to belowground biomass ratios, above- to fine root biomass ratios, and fine root to rhizome ratios were compared between the chronic low-level N addition treatment and control, and between sandy-loam and clay-loam using separate non-parametric Kruskal-Wallis tests.

For the separate, single year, high-level factorial N and P addition experiment, we tested for effects of each nutrient separately and in combination across the two soil texture categories on total aboveground biomass using a three-way ANOVA. Prior to that analysis, the data were visually and statistically inspected to confirm the assumptions of normality and homogeneity of variance using the above-mentioned statistical packages.

Plant Community Aboveground Responses to the Chronic Low-level Nitrogen Addition Treatment

Neither plant community productivity nor species richness or diversity were significantly altered by 16 years of annual low-level N additions across a hayfield of varying clay concentration (6% - 41%, Table S1, p = 0.11, p = 0.28, and p = 0.35, respectively, Table S2), and there was no significant interaction between WFPS and added N with either of these variables (p = 0.24, p = 0.73, and p = 0.54, respectively, Table S2). Furthermore, the N addition treatment had no significant effects on either ammonium, nitrate, or phosphate availability to roots in mid-July as measured by the IEMs (p = 0.39, p = 0.18, and p = 0.35, respectively, Table S2). However, both the ammonium and nitrate (but not phosphate) fluxes decreased significantly with increasing soil WFPS across the field (p < 0.00001 and p < 0.00001, respectively, Table 1), explaining 68% and 54% of the variability in the nutrient fluxes, respectively.

Table 1 Model parameters used for chosen best-fit models for each response variable measured in relation to the chronic low-level N addition treatment, variation in soil clay concentration, and relevant environmental variables. Models presented here represent the best-fit models based off AICc selection criteria. The best fit model for total aboveground biomass included soil temperature along with water-filled pore space, while the best fit models for the other four variables contained only water-filled pore space. R² for WFPS-only models is presented, along with the overall R² for the WFPS plus soil temperature aboveground biomass model.

Above- and Belowground Biomass Responses to Field Variation in Soil Texture and its Interaction with Soil Moisture

Plant community aboveground biomass was clearly positively correlated with field variation in soil clay concentration over the range from 6% up to a peak of ~15% clay content, and then remained generally consistent at higher clay concentrations (Fig. 2a). Likewise, community aboveground biomass increased with mean growing season soil WFPS up to a peak of ~15% and then remained generally high (Fig. 2b). Overall, in terms of the study site’s experimental layout, soil clay concentrations were on average >3 times larger in the plots on the clay-loam section of the field compared to those on the sandy-loam (Tables 2 and S1), and this textural difference was associated with >2 times greater mean growing season soil moisture and soil WFPS, and ~1.5 times more aboveground biomass (Table 2). Correspondingly, our statistical model analysis indicated that total aboveground biomass across the field increased significantly with rising mean growing season WFPS (p = 0.0008, Fig. 2b, Table 1) and decreased significantly with rising mean soil temperature (p = 0.012, Fig. S4, Table 1). In summary, spatial variability in WFPS across the field was the most important variable influencing aboveground biomass and explained 51% of the observed variability, while the combination of it and soil temperature explained 62% of the shoot biomass variation across our study site (Table 1). Furthermore, mean growing season soil moisture also increased with rising clay concentrations up to an asymptote of ~15% clay content, indicating an important interaction between soil pore space (which is determined by texture) and soil water availability (Fig. 2c). Together, these consistent asymptotic relationships, and the statistical model analysis, indicate that total plant community aboveground biomass was much more influenced by variation in soil water availability across the relatively dry, low clay content plots of the sandy-loam section of the field compared to the clearly moister, clay-rich plots of the clay-loam section (See Methods and Table 2).

Table 2 Mean values of total plant community aboveground biomass, total belowground biomass and its individual components, species richness, species diversity (Shannon-Weiner Diversity Index), soil clay and sand concentrations, soil bulk density, mean growing season soil water-filled pore space (WFPS), soil moisture (volumetric water content), soil temperature, and mid-July soil nutrient fluxes for the control and chronic low-level N addition treatment plots (n = 6, standard errors in parentheses) in the clay-loam and sandy-loam sections of Stoke’s hayfield experimental site.

In contrast to total aboveground biomass which ranged from ~180 to ~600 g m^-2 (Fig. 2a), total belowground biomass was generally larger and more variable, ranging from 220 g m^-2 to ~1000 g m^-2 (Table 2, Fig. S5a). Neither total belowground biomass, nor its components (i.e. rhizomes and fine roots), differed significantly between the low-level N addition and control plots (Total Belowground: p = 0.43, Rhizome: p = 0.54, Fine Root: p = 0.42, Table S2, Fig. S5), or in relation to spatial variation in WFPS (Total Belowground: p = 0.69, Rhizome: p =0.77, Fine Root: p = 0.66, Table S2, Fig. S5). Accordingly, since the shoot biomass was larger on the clay-loam, the mean ratio of plant community aboveground to fine root biomass was significantly higher there (2.32) compared to the sandy-loam section of the field (1.45) (p = 0.009, Table S3). By contrast, the ratio of rhizome to fine root biomass ranged from 0.48 to 1.28 but did not differ significantly between N addition and control (p = 0.6, Table S3) or soil texture categories (p = 0.7, Table S3).

Species Richness and Diversity Index Responses to Environmental Variables

The total species count across all plots on the clay-loam section was 50% higher (30-31 species) than on the sandy-loam section (19-20 species), and mean species richness per plot was twice as high (17 on the clay-loam compared to 8-9 species on the sandy-loam, Table 2). However, just as for biomass, there was absolutely no impact of the 16 years of chronic low-level N additions (Tables 2 and S2, Fig. 3a). Plot species richness increased significantly with rising mean growing season WFPS (p = 0.0001, Table 1), with the statistical model suggesting that WFPS explained 80% of the variation in species richness across the field (Fig. 3a).

The Shannon Diversity Index values ranged from 1.2 to 2.25 across the field (Fig. 3b), and again were higher on the clay-loam and not affected by the low-level N addition treatment (Tables 2 and S2, Fig. 3b). Likewise, the Shannon Diversity Index varied significantly with plot differences in WFPS (p = 0.0001, Table 1), which explained 47% of the variation in species diversity across the field (Fig. 3b).

Relative abundance graphs for the overall plant communities in the N addition and control plots on the predominantly clay-rich and sand-rich soil sections of the field (i.e., n = 6 for each category) illustrated significant differences in species composition and relative proportions (evenness). Smooth Brome (Bromus inermis) was the dominant species on the clay-loam, comprising 27% and 36% of the total biomass in the control plots and N addition plots, respectively (Fig. 4a, b). By contrast, Timothy Grass (Phleum pratense) was the dominant species on the sandy-loam and made up 35% and 29% in the control and N addition plots, respectively (Fig. 4c, d). Red Clover (Trifolium pratense) was the only forb (and N-fixing) species present at high proportions in the field and accounted for approximately 13% and 7% of the total biomass on the clay-loam soils in both the control and N addition plots but was absent from the sandy-loam plots (Fig. 4). Despite the high species richness across both soil textures, most species were present at very low proportions (< 5%), and the top five species on each soil texture category and treatment accounted for roughly 80% of the total biomass.

Effects of the High-Level Factorial Nitrogen and Phosphorus Additions on Aboveground Biomass

In the separate, single season experiment to directly test whether total plant community growth was primarily limited by nutrient availability, neither the high-level additions of N alone, P alone, nor their combination, significantly enhanced community aboveground biomass (N: p = 0.50, P: p = 0.76, N+P: p = 0.73, Table 3). Nevertheless, the three-way ANOVA indicated that total aboveground biomass was significantly higher on the clay-loam compared to the sandy-loam (p < 0.0001, Table 3), and that there were no significant interactions between soil texture and the nutrient treatments. These results are consistent with the lack of response to the low-level N addition treatment described earlier, together indicating that soil textural variation was a much stronger determinant of aboveground production than either N and/or P supply.

Table 3 Mean total plant community aboveground biomass (g dry mass m^-2) in response to the separate single growing season factorial high-level fertilizer addition treatments (Nitrogen, Phosphorus, Nitrogen and Phosphorus) on the predominantly clay-loam and sandy-loam sections of Stoke’s Field (n = 6, standard errors in parentheses). The high-level fertilizer additions were made in late Spring 2020 and the aboveground biomass was harvested ten weeks later.

Agroecosystem Production did not Respond to the Chronic Low-Level N Addition or to the Single High-Level N and P Fertilization Treatments

The annual low N additions for 16 years had no significant effects on either above- or belowground productivity, species richness, species diversity, or soil solution nutrient fluxes, refuting the N availability-related components of hypotheses 1, 2, and 3. However, all the aboveground plant community variables were significantly larger on the clay-loam plots compared to the sandy-loam, supporting the hypothesised influences of variation in soil texture. Previous research at Stoke’s Field hay grassland in 2014 observed a similar lack of response in aboveground biomass production after nine years of simulated atmospheric N deposition enhancement, and a similar strong soil texture effect (Constant and Grogan, Unpublished results).

Aboveground hay production at our site may have been unable to respond to enhanced N because growth was co-limited by N and P (Fay et al. 2015). Furthermore, previous literature has shown that adding N alone can have increasing, decreasing, and no effect on species richness (Bowman et al. 2012; Song et al. 2011), while N in combination with other nutrients (often P and K) can cause rapid declines in the same metrics (Midolo et al. 2019; Suding et al. 2005). To rule out the possibility of co-limitation as an explanation, we therefore established a separate, factorial high-level N and P fertilization experiment in the Spring of 2020, on both the clay-loam and sandy-loam soil sections of Stoke’s hayfield. Contrary to our hypothesis 4, although aboveground biomass was substantially larger on the clay-loam overall, it did not respond to any of the high-level fertilizer addition treatments on either soil texture category, strongly suggesting that aboveground plant community production across Stoke’s hayfield over the 2020 growing season was not limited by either N or P, nor was there co-limitation. Together, these results from separate experiments in different years at our study site consistently indicate that plant community shoot production, species richness, and species diversity are generally larger on the clay-loam compared to the sandy-loam sections of this semi-natural grassland agroecosystem (Tables 2 and 3), and that experimentally enhanced nutrient availability has negligible impacts.

This lack of grassland productivity response to nutrient enhancement is not uncommon. For example, in the global scale experimental study of nutrient limitation on grassland productivity across 5 continents, although the majority of the 42 tested sites demonstrated some fertilization response, more than ¼ of all sites did not respond to additions of either N, P, K and micronutrients, or any combination thereof (Fay et al. 2015). Site-specific climate, soil development age, and innate soil fertility were broadly posited as possible explanations. These latter factors can also interact with experimental nutrient addition to determine the actual supply of plant-accessible soil nutrients. For example, Bai et al. (2010) reported that the highest plant growth responses to N addition in a Eurasian steppe grassland occurred during years of high precipitation and hypothesized that the variation in responses of the aboveground biomass stemmed from differences in plant-soil feedbacks that influenced water availability.

For our site, we suggest that severe soil moisture limitation on plant community growth at least partly explains the lack of nutrient addition effects in the long-term low-level N addition study and in the separate one-year factorial high-level N and P addition study. Precipitation levels over the growing season in southern Ontario are generally low and often sporadic (primarily via occasional storms, and therefore the plant communities on both the clay-loam and sandy-loam sections of the field must be evolutionarily well-adapted to low soil water availabilities. Furthermore, in the harvest year for both experiments (i.e., 2020), rainfall for May (when the fertilizer was added) was unusually low and followed by a below-average June compared to the climate records for the previous 40 years (Fig. 5a-c, Environment Canada 2021). Corresponding mean monthly air temperatures were low for May and average for June, but July was particularly hot (Fig. 5d-f), potentially driving substantial evapotranspiration losses of moisture from the soil that may have exacerbated soil water limitation to the vegetation toward the end of the growing season.

Furthermore, our aboveground biomass results are corroborated by those ofSerafini et al. (2019) from a nearby sandy-loam old field meadow experiment with factorial high-level fertiliser (N-P-K) and summer watering treatments (two levels: rainout shelters and weekly water additions). After 5 years of the manipulations, plant community biomass in the experimental drought conditions of the rain-out shelters was reduced by ~ 35% and was unresponsive to the fertilizer amendment, suggesting that plant growth in those plots was severely and primarily limited by soil water availability. By contrast, growth was enhanced by the fertiliser in both the ambient and water addition plots but only by ~ 20%, and there was no significant interaction (i.e., fertilization did not significantly enhance production in the water-added plots compared to the ambient moisture plots). To explain the latter result, the authors hypothesized that the sandy-loam texture of the site may have caused the added water to quickly drain down through the soil profile, restricting the duration of elevated soil moisture conditions and therefore restricting the potential for a plant growth response. In summary, the Serafini et al. (2019) study provides some evidence suggesting that semi-natural grassland plant community production in this region is at best only moderately responsive to chronic high-level nutrient enhancement and only slightly responsive to water additions. By contrast, the rainout shelter responses provide clear evidence that productivity and community structure are highly sensitive to drought. Accordingly, we propose that growth responses to fertiliser are generally small in these grasslands, and that the particularly dry conditions in the 2020 harvest year compared to the long-term climate record (described above) probably contributed to the absence of nutrient addition effects in both the chronic low-level N addition and single year high-level N + P addition experiments reported here.

In addition, several other factors may have contributed to the absence of fertiliser effects at our site. Some of the added N may have been lost via leaching and denitrification, although the generally mesic-dry soil conditions would likely restrict such processes. The soil microbial biomass may have accumulated and sequestered some of the added nutrients making them unavailable to the plants, although measurements of microbial biomass N (and carbon) 3 years after the beginning of the experiment indicated no such effect (Buckeridge and Grogan, Unpublished results). In any event, consistent observations over multiple years of greener shoots in the low-level N addition plots within 1–2 weeks of its addition each Spring (Fig. S6) indicate that the plants were able to rapidly acquire and translocate at least some of the added N, supporting our proposition that subsequent water scarcity later in the growing season may have been a primary constraint on the vegetation’s potential to use that acquired N for enhanced growth.

Interaction Between Soil Texture and Soil Moisture (WFPS) Determines Aboveground Biomass Productivity

Mean growing season soil water-filled pore space (WFPS) was consistently the most powerful explanatory variable in our models (Table 1), suggesting that variation in soil water availability to plants was the primary determinant of patterns of aboveground productivity, species richness, and Shannon diversity across the hayfield. For example, aboveground biomass was related to WFPS by an asymptotic relationship (Fig. 2b), with WFPS alone explaining 51% of its total variation (Table 1). Mean growing season WFPS was very closely correlated with soil moisture (Fig. S7), and examination of the individual plot soil moisture data clearly indicates strong positive relationships with clay concentration across the full range from relatively dry and wet sampling days through the growing season (Fig. S8). Therefore, soil clay concentration clearly greatly influenced WFPS. However, although the relationship between WFPS and absolute soil water content was broadly linear (Figs. 2c and S8), theory would suggest that its relationship to plant-accessible water was unimodal because of matric potential, a component of water potential referring to the direct adsorption of water to mineral soil particles (Brady and Weil 2008). Sandy soils with little clay content have low matric potential, but also store relatively little water. By contrast, although finer-textured soils have a larger water holding capacity, the high matric potential associated with the relatively large mineral surface area in clay-rich soils means that a substantial amount of the stored water is unavailable, making it difficult for plants to access water when those soils are mesic or relatively dry (Chapin et al. 2012). Therefore, plant-accessible water is determined largely by soil texture, with both sand-rich and clay-rich soils having relatively low plant available water as a result of either poor water retention or excessive adhesion of the water to the soil particles (O’Geen 2013).

In our study, aboveground biomass increased rapidly with rising WFPS in the low clay concentration plots (Fig. 2a, b) indicating a strong productivity response to even small increases in soil water availability to plants in those relatively dry sandy plots. By contrast, at plot clay concentrations > 15%, aboveground biomass fluctuated somewhat but generally appeared independent of WFPS (Fig. 2b), suggesting that productivity was not enhanced above the 15% clay threshold because there was no corresponding increase in plant-available water. Accordingly, in the context of matric potential, we conclude that the general lack of aboveground biomass response to rising clay concentrations beyond the ~ 15% threshold at our site is likely due to declining plant-accessible water due to increasing soil clay-water molecule binding (Fig. 2). Our study is interesting in that the textural variation at Stoke’s hayfield is fairly modest – all plots have soils that fall within the broad ‘loam’ category, and yet variation even within this category (i.e., clay-loam compared to sandy-loam) clearly had profound impacts on plant production and community structure.

Interaction Between Soil Texture and Soil Moisture (WFPS) Determines Plant Species Diversity

Plant species richness per plot was twice as high on the clay-loam compared to the sandy-loam soils, and WFPS explained ~ 80% of its total variation across the field (Tables 1 and 2, Fig. 3a). Individual plot average soil water content on four of the five sampling dates during the 2020 growing season ranged from just 0.4–18% in the low clay content soils, and 4–31% in the more clay-rich soils (Fig. S8). By contrast, even on the one relatively wet measurement day (July 15), soil moisture ranged from 9–18% in the low clay content soils, and 23–31% in the more clay-rich soils (Fig. S8). We conclude that just as observed in other mesic/semi-arid grasslands (Wu et al. 2014), it is likely that the severe water stress caused by the generally extremely dry soil conditions on the sandy-loam precluded many species from establishing and surviving on this portion of the field.

The very strong interconnected influences of soil clay concentration and WFPS on species richness were clearly the primary determinant of the patterns in the Shannon-Weiner Diversity Index. Overall, the plant community structure across the field was characterized by low evenness and the consistent dominance of a few grass species. Rank abundance plots confirmed that the species diversity response to plot differences in WFPS was primarily related to changes in species richness rather than evenness (Fig. 4). Dominant grass species such as Smooth Brome (Bromis inermis), Timothy Grass (Phleum pratense), and Tall Fescue (Lolium arundinaceum) accounted for about 80% of the total aboveground biomass on both soil texture categories. The consistent dominance of these few introduced grasses over native forbs and grasses across a range of soil clay concentrations (and therefore a range of water availability) at Stoke’s hayfield provides important in situ plant community evidence to support greenhouse plot studies suggesting these species have a strong competitive advantage that will persist in the drier soils expected under future climate change in North America (e.g., Duell et al. 2021). This conclusion, along with the lack of N addition treatment effects reported above, means farmers can anticipate that hay species composition will continue to be dominated by these introduced species even as future atmospheric N deposition rates continue to rise.

Root and Rhizome Biomass did not Respond to Nitrogen Addition Treatment or Correlate with any Environmental Parameter Measured

Neither total belowground biomass, nor its components (fine roots and rhizomes) responded to the N addition treatment, nor were they correlated with mean growing season WFPS or soil temperature. To date, relatively few grassland studies have included both the above- and belowground biomass to assess a more complete picture of plant community structure and function (Kobiela et al. 2016). Although our soil samples were unusually large (~ 30 x 30 x 10 cm depth), and we took great care to quantify these variables carefully (Protocol S1), there was clearly large variability among individual plots (Fig. S5). Nevertheless, the mean ratio of plant community aboveground to fine root biomass was significantly higher in the clay-loam (2.32) compared to the sandy-loam section of the field (1.45), (with similar patterns for total belowground and rhizome biomass – Tables 2 and S3) suggesting that in terms of proportional allocation, plant investment in root production was relatively high in the latter, presumably to optimise water uptake in those generally very dry soils.

The consistent lack of response to the chronic low-level N addition treatment suggests that future increases in atmospheric N deposition are unlikely to have major impacts on hay productivity and diversity in Ontario’s mesic semi-natural grasslands. By contrast, we observed strong interconnected influences of moderate variation in texture within the ‘loamy’ soils of our study site and soil moisture (i.e., WFPS) on multiple plant community-level properties. Soil moisture in this region is expected to become more limiting in the future(IPCC 2021) as a result of anticipated increases in summer temperatures and evapotranspiration losses and decreases in summer precipitation. From a farming perspective, our results suggest that the impacts of future climate on hay production and species composition will be highly spatially variable because of critical differences in water stress caused by even moderate variation in soil texture (i.e., within the range from clay-loam to sandy-loam). Furthermore, from an agroecosystem biodiversity perspective, we conclude that understanding the influence of spatial heterogeneity in soil texture on plant community responses will be critical to managing and maintaining the species conservation value of mesic natural and semi-natural grasslands.

nitrogen (N); carbon dioxide (CO₂); phosphorus (P); potassium (K); urea (CO(NH₂)₂); ion exchange membranes (IEM); cation exchange membranes (CEM); anion exchange membranes (AEM); water-filled pore space (WFPS); soil volumetric water content (VWC); soil bulk density (BD); particle density (PD); Akaike’s information criterion (AIC).

ACKNOWLEDGMENTS:

This research was conducted in the area that is now known as Kingston, Ontario, which is the traditional territory of the Anishinaabek and Haudenosaunee Peoples. We are deeply honoured to live, learn, and play on these lands. We thank Drs. Neal Scott, Stephen Lougheed, and Christopher Eckert for their comments and insights on the thesis that gave rise to this manuscript. Thanks also to Sarah Gordon, Kayleigh Casmey, Mallory Sirimanne, and Kieran Mcfarlane for help with field and lab work. Specific measures were taken by the authors to reduce the environmental impacts of the data collection for this scientific study (Table S4).

STATEMENTS AND DECLARATIONS:

Funding: Funding was provided by a National Science and Engineering Research Council (NSERC) Discovery Grant (NSERC RGPIN 262750) held by Dr. Grogan.

Competing Interests: The authors have no competing interests to declare that are relevant to the content of this article.

Authorship Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Meghan Hamp. The first draft of the manuscript was written by Meghan Hamp and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability: Data are available online through Zenodo. DOI 10.5281/zenodo.8299616

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Plant production and community structure in a mesic semi-natural grassland: Moderate soil texture variation has a much stronger influence than experimentally increased atmospheric nitrogen deposition

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Version 1

Abstract

Aims

Methods

Results

Conclusions

Figures

INTRODUCTION

METHODOLOGY

Stoke’s Field Hay Grassland Site Characteristics and History

Atmospheric Nitrogen Deposition Experimental Setup

Above- and Belowground Plant Collection and Mass Analysis

Belowground Biomass Sample Processing

Ion Exchange Membrane Measurement of Soil Nitrogen and Phosphorus Availability to Roots

Abiotic Measurements

Soil Texture Calculation and Classification

Soil Texture and Soil Moisture Interaction (Water Filled Pore Space)

Nitrogen And Phosphorus Growth Co-Limitation Study

Statistical Analyses

RESULTS

DISCUSSION

Interaction Between Soil Texture and Soil Moisture (WFPS) Determines Aboveground Biomass Productivity

Interaction Between Soil Texture and Soil Moisture (WFPS) Determines Plant Species Diversity

CONCLUSIONS

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1