Modification by earthworms of effects of soil heterogeneity and root foraging in eight species of grass

Journal Pre-proofs Modification by earthworms of effects of soil heterogeneity and root foraging in eight species of grass Lu Liu, Peter Alpert, Bi-Cheng Dong, Fei-Hai Yu PII: DOI: Reference:

S0048-9697(19)34933-2 https://doi.org/10.1016/j.scitotenv.2019.134941 STOTEN 134941

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Science of the Total Environment

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Please cite this article as: L. Liu, P. Alpert, B-C. Dong, F-H. Yu, Modification by earthworms of effects of soil heterogeneity and root foraging in eight species of grass, Science of the Total Environment (2019), doi: https:// doi.org/10.1016/j.scitotenv.2019.134941

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Modification by earthworms of effects of soil heterogeneity and root foraging in eight species of grass

Lu Liu1,2,3, Peter Alpert4, Bi-Cheng Dong3, and Fei-Hai Yu1,3*

1

Institute of Wetland Ecology & Clone Ecology/ Zhejiang Provincial Key Laboratory

of Plant Evolutionary Ecology and Conservation, Taizhou University, Taizhou 318000, China 2

School of Water Conservancy, North China University of Water Resources and

Electric Power, Zhengzhou 450045, China 3

School of Nature Conservation, Beijing Forestry University, Beijing 100083, China

4

Biology Department, University of Massachusetts, Amherst, Massachusetts 01003,

USA

*

Corresponding author. Email: [email protected]

Short title: Nutrient heterogeneity, earthworms, and root foraging

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ABSTRACT Spatial heterogeneity of soil nutrients and earthworm activity can each increase the performance of plant species, but their interactive effects have been little studied. The ability of plants to forage for nutrients by concentrating roots where nutrients are concentrated can partly explain the positive effects of nutrient heterogeneity, but whether root foraging can help explain the positive effects of earthworm activity is untested. We conducted a greenhouse experiment in which we grew eight species of Poaceae in homogeneous and heterogeneous soils with or without the earthworms Eisenia fetida and Metaphire guillelmi and measured net accumulation of plant mass and tillers. Effects of heterogeneity and earthworms on plant performance were positive in most species. The presence of earthworms reduced the directly measured effect of heterogeneity on total mass in some grass species. Most species showed root foraging ability. Ability showed no relationship to effects of heterogeneity or earthworms on final total dry mass. However, earthworms reduced foraging in some species, possibly by lessening heterogeneity. Earthworm activity in heterogeneous soil may thus reduce the benefits of root foraging for nutrients in plants.

Keywords: detritivory, Eisenia fetida, Metaphire guillelmi, nutrient heterogeneity, Poaceae, root foraging ability

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Introduction Fine-scale spatial heterogeneity in soil nutrient availability is common in natural habitats and can strongly affect plant growth (Jackson & Caldwell, 1993; Day et al., 2003). Increasing nutrient heterogeneity can increase growth even when total nutrient availability is held constant, showing that heterogeneity itself is important. One reason for this positive effect of heterogeneity is likely that plants often concentrate roots where nutrient availability is relatively high (Jackson et al., 1990; Kembel & Cahill, 2005; García‐Palacios et al., 2012), a response often referred to as nutrient or root foraging (Wijesinghe et al., 2001; Kembel & Cahill, 2005; de Kroon et al., 2009). Species differ in root foraging ability, and species with greater ability tend to show greater positive effects of soil nutrient heterogeneity on growth (Einsmann et al., 1999; Fransen et al., 2001; Bliss et al., 2002; Xue et al., 2018). Earthworm activity is another common factor in soils that strongly affects plant growth (Callaham et al., 2001; Blouin et al., 2005; Crumsey et al., 2015). For example, movement of earthworms through the soil changes permeability and infiltration, and provides paths of reduced resistance to root growth (Jégou et al., 2000; Don et al., 2008). Earthworms excrete hormone-like chemicals that affect plant growth or break seed dormancy (Ayanlaja et al., 2001). Detritivory by earthworms can increase mineralization of nitrogen by microbes, redistribute organic matter, and aggregate soil (Bottinelli et al., 2015; Angst et al., 2017; Frouz, 2017). Amounts of

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amino acids and nitrogen tend to be relatively high in soil adjacent to earthworm burrows (Hoang et al., 2016). Effects of nutrient heterogeneity and of earthworms on plants are likely to interact. For instance, the degree to which earthworm activity affects plants depends on other soil properties, such as texture and concentrations of nutrients and organic matter (Brown et al., 2004). The earthworm Lumbricus terrestris can promote heterogeneity in texture by forming soil aggregates (Shuster et al., 2001). Earthworms might also increase nutrient heterogeneity in relatively homogeneous soils by concentrating nutrients in excretions but decrease heterogeneity in relatively heterogeneous soils by consuming nutrients in high-nutrient patches and excreting them in low-nutrient patches. Few studies have tested for interactive effects of earthworms and nutrient heterogeneity on the growth of plants. Liu et al. (2017) reported that earthworm activity did not modify the positive effects of soil nutrient heterogeneity on Hydrocotyle vulgaris. Wurst et al. (2003) found that patchy distribution of litter promoted root growth in Plantago lanceolata only in the presence of earthworms. No previous work appears to have tested whether effects of earthworms on plants are related to root foraging ability. To more extensively test for interactions between the effects of soil nutrient heterogeneity and earthworm activity and to test whether these effects are related to root foraging, we conducted a greenhouse experiment with eight species of grass

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(Poaceae). We crossed the presence and absence of two common earthworms, one epigeic and one anecic, with the presence and absence of soil nutrient heterogeneity, keeping total soil nutrient availability constant. We used plant species from the same family to reduce differences between species other than foraging ability, and used grasses because earthworms are known to affect their growth (Wurst et al., 2005; Laossi et al., 2009). Based on previous studies on earthworm activity and nutrient heterogeneity, we hypothesized that plants would grow more (1) when earthworms were present than when they were absent and (2) when soil nutrient availability was heterogenous than when it was homogeneous. Based on previous work on root foraging in plants, we hypothesized (3) that species with greater root foraging ability will show a greater positive effect of nutrient heterogeneity on growth. Based on the logical supposition that earthworm activity is likely to increase heterogeneity in completely homogeneous soil and to decrease heterogeneity in highly heterogenous soil, we hypothesized that (4) earthworms will increase the benefits of foraging ability when nutrient availability is highly homogeneous and decrease these benefits when nutrient availability is highly heterogeneous.

Materials and methods Species We selected eight grass species that are widespread in China, represent a range of belowground architecture and so possibly differ in foraging ability, and for which seed

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was available (Table 1). Seven of the species are perennials; one is an annual. Six of the species tend to root mainly at the base of a cluster of tillers; two species root mainly along stolons or rhizomes. Three of the species produce both stolons and rhizomes, two produce only stolons, one produces only rhizomes and two usually produce neither stolons or rhizomes. Seeds were purchased from the Agricultural Institute of Jiangsu Province, China, and germinated in twice-washed river sand. Once seedlings reached a height of about 3 cm, 24 similarly sized individuals of each species were selected for use. Eisenia fetida Savigny (Lumbricidae, epigeic redworm) is one of the most common earthworms in the world (Gunadi & Edwards, 2003; Aira et al., 2006). It is native to Europe but has been introduced to every other continent except Antarctica. Individuals are about 6 to 8 cm long and live in the upper soil. Metaphire guillelmi Michaelsen (Megascolecidae, anecic grayworm), is a common earthworm that lives primarily in semi-permanent, vertical burrows below the upper soil, though it forages for coarse organic litter at the soil surface (Sims & Easton, 1972). M. guillelmi is widespread in many provinces of China, including Hebei, Shandong, Jiangsu, Zhejiang, Guangdong, and Guangxi. Individuals are about 7 to 15 cm long and grow rapidly until sexually mature. The main reason for including both an epigeic and an anecic species of earthworm was to ensure activity over a relatively wide range of soil depths. The two species might reduce each other’s activity somewhat due to overlap in soil depths and thus competition for soil resources but likely also complement each

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other’s effect on soils by concentrating their activity at different depths. In China, both species are widespread in commercial composting sites, semi-natural grassland, and arable land (Cao et al., 2006; Tiunov et al., 2006), and have been frequently used in studies on soil remediation, cultivation, and enzymatic activity (Spurgeon & Hopkin, 1999; Zhang et al., 2000; Aira et al., 2006; Chen et al., 2017). Earthworms were collected from Jurong, Jiangsu Province, China (31.9°N, 119.2°E), and maintained for two weeks before the experiment in a container (55 × 45 × 35 cm) filled with the loamy soil used in the experiment.

Experimental design The experiment used a fully factorial design with six replicates of 32 treatment combinations, the eight grass species crossed with two earthworm treatments (present or absent) crossed with two soil treatments (homogeneous or heterogeneous). For the heterogeneous soil treatment, circular plastic pots that were 16 cm in diameter at the top, 12 cm in diameter at the bottom, and 15 cm tall with 0.5-mm mesh in the bottom were divided into four equal quadrants with plastic partitions. Two opposite quadrants of each pot were each filled with 500 ml of an 8:2 mixture of nutrient-poor loam and commercial potting soil to create low-nutrient patches. The other two quadrats were each filled with 500 ml of a 2:8 mixture of the loam and potting soil to create highnutrient patches. The low-nutrient patch contained 0.209% N, 0.115% P, and 3.101% organic C; the high-nutrient patch contained 0.598% N, 0.104% P, and 10.367%

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organic C, as measured in samples sent to the Institute of Botany, Chinese Academy of Sciences. The loam (0.128% N, 0.138% P and 1.169% organic C) was collected 10-50 cm below the surface of uncultivated land in Huangyan, Taizhou, Zhejiang Province, China (28.6°N, 121.1°E), on 15 July 2017 and sieved through a 1-cm mesh. The potting soil was purchased from Meishimei Bio-Tech Co. Ltd, Beijing, China (65% organic peat soil, 16% vermiculite, 12% fully-fermented straw compost, and 2% pearlite, and 5% other components). The partitions between quadrants were removed after filling the pot. For the homogeneous soil treatment, similar pots were filled in the same way, but the soil was thoroughly mixed after the partitions were removed. Total amounts of soil nutrients per pot were thus the same in both soil treatments. Manipulating soil heterogeneity by varying proportions in soil mixtures is likely to be relatively realistic and appropriate for tests of effects of earthworms on plant growth in different soils (Wijesinghe et al., 2001; Bamminger et al., 2014; Keser et al., 2014; Wang et al., 2017). Pots were arranged haphazardly in a single array on a bench in the greenhouse of the Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation at Taizhou University, Taizhou, Zhejiang Province, China. On 25 September 2017, a single seedling was planted in the center of each pot and allowed to establish for 12 days. Half of the 24 seedlings of each species were haphazardly assigned to each soil treatment, and 12 pots of each soil treatment were haphazardly assigned to receive each species. Half of the pots in each soil treatment

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containing each species were haphazardly assigned to receive three earthworms (2 individuals of E. fetida and 1 individual of M. guillelmi), which were themselves haphazardly assigned to plant species. This provided a density of about 150 earthworms/m2, slightly higher than the range of common densities in arable soil but lower than densities found in some coniferous forests (Smith et al., 2008). The initial length of the earthworms was 6.21 ± 0.16 cm (mean ± SE, n = 20). The initial weight of the earthworms was approximately 0.5 and 2.5 g. Earthworms were added on 7 October and treatments continued for 52 days. Daytime and nighttime temperatures were set to 28°C and 18°C respectively, relative humidity to 65%, and daylength to 15 hours, following Wurst et al. (2004). Enough tap water was added every three to five days to keep the soil moist. On 27 November, the plant in each pot was divided into aboveground parts, belowground parts from high-nutrient quadrats, and belowground parts from lownutrient quadrants. In the homogeneous treatment, where all quadrats were mediumnutrient, two opposite quadrants were haphazardly assigned to be compared to the high-nutrient and the remaining two quadrants to be compared with the low-nutrient quadrats in the heterogeneous treatment. Plant parts were dried at 75 °C for 48 hours and weighed. A few replicates of some grass species in some treatments, 10 plants in total, died before harvest.

Data analysis

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Two-way ANOVAs were used to test the effects of earthworms (present or absent) and soil heterogeneity (homogeneous or heterogeneous) on the final number of tillers and final dry total, aboveground, and belowground mass of each plant species separately. A four-way ANOVA was used to test the effects of grass species, earthworms, soil heterogeneity, and quadrant (high- or low-nutrient, repeated measure) on the final dry belowground mass of plants. All effects were treated as fixed. To check whether it would change results to consider grass species as a random factor, we also ran a threeway ANOVA of effects of earthworms and heterogeneity as fixed factors and species as a random factor (Supplementary Materials); results were little changed. Analyses were conducted using SPSS 22.0 (SPSS, Chicago, IL, USA). To test for relationships between foraging ability and effects of soil heterogeneity and presence of earthworms, ability and effects were quantified for each species as follows. Foraging ability was calculated as proportion of dry belowground mass located in the high-nutrient quadrants in the heterogeneous treatment. Effect of soil heterogeneity was calculated as mean total dry mass in the heterogeneous treatment without earthworms minus mean total mass in the homogeneous treatment without earthworms divided by mean total mass in the latter treatment. Effect of earthworms on effect of soil heterogeneity was calculated as this value subtracted from the analogous value for treatments with earthworms. Each of these two effects as well as effect of earthworms on total mass in heterogeneous soil (mass in heterogeneous soil with earthworms minus mass in heterogeneous soil without earthworms divided by

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mass in the latter treatment) and in homogeneous soil (analogous value for homogeneous soil) was then regressed on species mean foraging ability.

Results Five of the eight grass species accumulated significantly more final total dry mass (P < 0.05) in the heterogeneous than in the homogeneous soil treatment (Fig. 1, Table 2). These were also the five species that were perennial and had clumped roots (Table 1). A different set of five of the eight grass species accumulated significantly more total mass with than without earthworms (Fig. 1, Table 2). No species accumulated more mass in homogeneous than in heterogeneous soils or without than with earthworms. Effects of soil heterogeneity and earthworms on total mass did not interact in any grass species as tested with ANOVAs (Fig. 1, Table 2: all P > 0.05). Effects of soil heterogeneity and earthworms on final dry aboveground mass, belowground mass, and final number of tillers per plant were generally qualitatively similar to effects on total mass (Supplementary Material). Effects on aboveground mass tended to be stronger than those on belowground mass and number of tillers (Table 2). These results exhibited that nutrient heterogeneity and earthworm activity can increase plant growth but provided no evidence for their interaction on plant growth. Across species, grasses had more final belowground dry mass in high- than in low-nutrient quadrants in the heterogeneous soil treatments, as would be expected if

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species foraged for nutrients via root growth (Fig. 2, Table 3). The difference between mass in high and low nutrients differed between species, providing evidence that species differed in foraging ability. Across species, effect of earthworms did not interact with effect of quadrant (Table 3: effect of quadrant x species x earthworms). However, some individual species did show an interactive effect of earthworms and quadrant. For example, belowground mass of Paspalum notatum was more than twice as great in high- as in low-nutrient quadrants without earthworms but only about 50% higher in high- than in low-nutrient quadrants with earthworms (Fig. 2). Foraging ability, as quantified by proportion of belowground dry mass located in the high-nutrient quadrants in the heterogeneous treatment without earthworms, differed significantly between species (F7,46 = 2.579; P = 0.029). Paspalum notatum showed significantly higher foraging ability than Elymus dahuricus (Tukey test, P = 0.05: Paspaluma Poaab Loliumab Zoysia matrellaab Agrostisab Festucaab Loliumab Zoysia tenuifoliaab Elymusb). All species except Elymus dahuricus and Poa annua showed significant foraging ability (t-tests for difference of mean proportion of belowground mass in high-nutrient quadrats from 0.5, P < 0.05). There was no evident positive relationship between belowground foraging ability and the effect of nutrient heterogeneity on final total dry mass in the absence of earthworms (Fig. 3A; linear regression of effect of heterogeneity on foraging ability: R2 = 0.05, P = 0.61). On the contrary, the species with the lowest foraging ability, Elymus dahuricus, showed twice as much effect of heterogeneity as any other species.

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Foraging ability likewise showed no relationship to the effect of earthworms on the effect of heterogeneity on total mass (Fig. 3B; R2 = 0.02, P = 0.73), which was near zero to negative, as might be expected if earthworms tended to reduce differences between high- and low-nutrient quadrants. There was no relationship between foraging ability and effect of earthworms in either homogeneous (Fig. 3C; R2 = 0.06, P = 0.57) or heterogenous soil (Fig. 3D; R2 < 0.01, P = 0.92).

Discussion Results supported the hypothesis that heterogeneity of nutrients promotes growth of plants. Spatial heterogeneity of nutrients increased net accumulation of total dry mass by 0 to 110% in the eight species of Poaceae tested even though the total amount of available nutrients was the same in heterogeneous and homogeneous soil treatments. This was consistent with many previous studies (e.g., Wijesinghe et al., 2001; Day et al., 2003; Maestre & Reynolds, 2007). Effect of nutrient heterogeneity did differ between species. For example, five species showed a significant positive effect of heterogeneity on final mass, and three showed no significant effect. This matched some differences between species in life history and architecture, since the five species with significant responses were also the five species that are both perennial and root mainly in a clump at the base of a cluster of culms. The three species that failed to show significantly higher mass in heterogeneous soil were the one annual and the two species that root mainly along

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stolons or rhizomes. This suggests that larger root masses may better exploit nutrient patches on the scale of individual plants. Results also supported the hypothesis that earthworm activity promotes growth in plants. Five of the eight species accumulated significantly more mass in treatments with than without earthworms, and no species showed a negative effect of presence of earthworms. Earthworms increased final total dry mass by 5 to 100% in different species. Many previous studies have likewise shown positive effects of earthworms on plant growth (Edwards & Bohlen, 1996; Scheu, 2003; Roubíčková et al., 2009; Noguera et al., 2010; Lv et al., 2016), including especially pronounced effects on grasses (Wurst et al., 2005; Laossi et al., 2009). Some studies have suggested that late-successional species benefit more earthworm activity than early-successional species (Roubíčková et al., 2009; Mudrák & Frouz, 2018), but there was no obvious relationship between species traits and effects of earthworms in this study. Results provided limited support for the hypothesis that effects of nutrient heterogeneity and earthworm activity on plant performance interact. ANOVAs of effects of heterogeneity and earthworms on total mass and number of tillers showed no significant interactive effects in any species. However, analysis of normalized differences between the mean total mass of species in treatments with and without heterogeneity indicated that the positive effect of heterogeneity was lower in the presence of earthworms in some species. Effects of earthworms on plants can clearly depend on soil characteristics. Previous studies have found that earthworms promoted

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plant growth more in sand than in clay, in low- than in high-nutrient soil, and in younger soil (Doube et al., 1997; Laossi et al., 2010; Ulrike et al., 2010). Interactions among different ecological groups of earthworms can structure earthworm communities in space, which could in turn influence the spatial variability of soil resources. For instance, E. fetida changes the spatial variability of resources near the soil surface by forming middens that may have relatively high concentrations of C and N. The burrows of M. guillelmi may concentrate nutrients and water deeper in the soil. No previous, published work appears to have tested specifically for interactive effects of earthworms and soil heterogeneity, and results here provide some initial indication that effects of soil nutrient heterogeneity on plant growth can depend upon earthworm activity. Future research that includes other functional groups of earthworms would help to extend this finding. For example, endogeic species might have strong effects on heterogeneity in deeper soil. Results did not support the hypothesis that species with greater nutrient foraging ability benefit more from nutrient heterogeneity. The positive effect of nutrient heterogeneity on plant growth is widely thought to be largely due to ability of plants to concentrate roots where nutrient levels are higher (Hutchings & John, 2004; Hodge, 2010; Gao et al., 2012; García‐Palacios et al., 2012). A number of previous studies have accordingly reported associations between foraging ability and promotion of growth by nutrient heterogeneity (Wijesinghe et al., 2001; Bliss et al., 2002; Mommer et al., 2012). We found no relationship between the mean foraging ability of the eight

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species tested and the normalized difference between their total mass in heterogeneous and homogeneous soil. Six of the species did concentrate their roots in high-nutrient soil, but only two species differed significantly in foraging ability, limiting power to detect a relationship. Nevertheless, results indicate that factors other than just placement of belowground mass determine how much plant species benefit from soil nutrient heterogeneity. Result did not support the hypothesis that earthworms increase the benefits of foraging ability when nutrient availability is highly homogeneous and decrease these benefits when nutrient availability is highly heterogeneous. Mean species foraging ability explained almost none of the variance in effect of earthworms on total mass of plants in either homogeneous or heterogeneous soil. However, earthworms did affect foraging in some species. ANOVA of belowground mass in different soil quadrants showed an interactive effect of species, quadrant, and earthworms, and one species had twice as great a difference between belowground mass in low- and high-nutrient quadrants in the absence as in the presence of earthworms. Earthworm activity is likely to decrease heterogeneity in highly heterogeneous soil both because earthworms can have greater positive effects on nutrient levels in soils with lower nutrient levels and because earthworms tend to move nutrients from high- to lownutrient areas (Rossi et al., 1997; Shuster et al., 2001; Araujo et al., 2004; RodriguezCampos et al., 2014; Knowles et al., 2016). If nutrient foraging takes place in response to nutrient heterogeneity, it is reasonable to expect that reduction in

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heterogeneity by earthworms could reduce foraging. Earthworms may thus reduce the benefits of ability to forage for nutrients, which could change the relative fitness of plant species that differ in foraging ability in habitats where soil nutrients are heterogeneous and earthworms are plentiful. In sum, results confirm previous work showing that soil nutrient heterogeneity and earthworm activity can increase plant growth and suggest that response to nutrient heterogeneity may be greater in plant species that tend to clump their roots. Results provide limited, initial evidence that earthworms can reduce the positive effects of nutrient heterogeneity in some plant species. It would be useful for future work to include longer-term studies on the possible interactive effects of earthworms and soil heterogeneity on plant growth (van Groenigen et al., 2014; Mudrák & Frouz, 2018). For example, introduced species of earthworms may be changing soil properties on a large scale (e.g., Tiunov et al., 2006), and such studies could help predict global changes due to invasive species.

Acknowledgments We thank Lu-Xi Chen for greenhouse management, and Drs. Qian Zhang and Fang-Li Luo for comments. Research was supported by the National Natural Science Foundation of China (Grants 31570413, 31870610 and 31761123001). The authors declare no conflicts of interest.

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Table 1 Species of Poaceae used in the study. Roots were either mainly in a relatively dense mass at the base of clusters of culms (clumped) or mainly dispersed along stolons or rhizomes (dispersed). Clonality indicates clonal growth by stolons (S), by rhizomes (R), or occasionally to rarely by stolons (s). Data are from the eFlora of China.

Species

Roots

Clonality

Life history

Agrostis stolonifera L.

clumped

S

perennial

Elymus dahuricus Turcz.

clumped

S

perennial

Festuca rubra L.

clumped

R

perennial

Lolium perenne L.

clumped

s

perennial

Paspalum notatum Flugge

clumped

R, S

perennial

Poa annua L.

clumped

s

annual

Zoysia matrella (L.) Merr.

dispersed

R, S

perennial

Zoysia tenuifolia Thiele

dispersed

R, S

perennial

25

Table 2 ANOVAs of effects of soil heterogeneity and earthworms on final dry mass and number of tillers in eight grass species. Symbols give P: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01; *** - <0.001.

Effect Agrostis stolonifera

df

Total mass

Heterogeneity (H) Earthworms (W) H×W Elymus dahuricus Heterogeneity (H) Earthworms (W) H×W Festuca rubra Heterogeneity (H) Earthworms (W) H×W Lolium perenne Heterogeneity (H) Earthworms (W) H×W Paspalum notatum Heterogeneity (H) Earthworms (W) H×W

1,19 1,19 1,19

4.84* 1.15ns 0.44ns

5.69* 2.54ns 0.86ns

3.23# 0.08ns 0.07ns

1.79ns 0.06ns 0.33ns

1,19 1,19 1,19

16.04** 1.93ns 0.86ns

23.38*** 2.36ns 1.61ns

8.77** 1.35ns 0.29ns

4.66* 0.02ns 1.45ns

1,19 1,19 1,19

5.53* 22.77*** < 0.01ns

8.72** 23.88*** 0.09ns

2.33ns 17.55*** 0.06ns

9.75** 21.4*** 1.22ns

1,16 1,16 1,16

15.43** 40.27*** 0.88ns

14.65** 40.06*** 0.05ns

8.77** 21.37*** 2.19ns

4.6* 37.38*** 0.33ns

1,20 1,20 1,20

9.92** 13.53** 0.01ns

12.48** 23.84*** 0.17ns

7.2* 6.5* 0.25ns

3.54# 10.56** 2.37ns

1,20 1,20 1,20

2.56ns 0.73ns 0.02ns

3.01# 2.73ns 0.03ns

1.6ns 0.08ns < 0.01ns

4.18# 1.19ns < 0.01ns

1,17 1,17 1,17

0.34ns 31.61*** 0.21ns

0.68ns 33.8*** 0.35ns

0.01ns 20.46*** 0.04ns

0.3ns 8.28** 0.15ns

1,20 1,20 1,20

2.41ns 5.48* < 0.01ns

5.19* 8.85** 0.03ns

0.69ns 2.76ns < 0.01ns

0.61ns 8.94** 0.61ns

Poa annua Heterogeneity (H) Earthworms (W) H×W Zoysia matrella Heterogeneity (H) Earthworms (W) H×W Zoysia tenuifolia Heterogeneity (H) Earthworms (W) H×W

Aboveground Belowground mass mass

26

Tiller number

Table 3 Repeated measures ANOVA of effects of grass species, soil heterogeneity, earthworms, and quadrant (high- or low-nutrient) on belowground mass. Symbols give P: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01; *** - <0.001.

Effect

df

F

Species (S)

7,150

27.66***

Heterogeneity (H)

1,150

20.35***

Earthworms (W)

1,150

40.35***

S×H

7,150

1.28 ns

S×W

7,150

2.82*

H×W

1,150

0.01 ns

S×H×W

7,150

0.45 ns

Quadrant (Q)

1,150

81.77***

Q×S

7,150

3.41**

Q×H

1,150

24.47***

Q×W

1,150

2.12 ns

Q×S×H

7,150

1.27 ns

Q×S×W

7,150

2.14*

Q×H×W

1,150

0.31 ns

Q×S×H×W

7,150

0.51 ns

Between-subject:

Within-subject:

27

Fig. 1 Effects of soil heterogeneity and earthworms on final total dry mass (mean + SE) in eight grass species. Symbols above pairs of bars show P (linear contrast based on ANOVA) that means for homogeneous and heterogeneous treatments did not differ within earthworm treatments: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01.

4 3 2 1

heterogeneous 5 Elymus dahuricus

homogeneous Agrostis stolonifera Total mass (g/plant)

Total mass (g/plant)

5

#

Total mass (g/plant)

Total mass (g/plant)

#

2 1 0

#

*

2 1 0

Lolium perenne

**

4 3 2

*

1

Poa annua

4 3 2 1 0 5

Zoysia matrella

4

Total mass (g/plant)

Total mass (g/plant)

5

1

5

Paspalum notatum

4 3

2

0

Total mass (g/plant)

Total mass (g/plant)

5

*

3

5

Festuca rubra

4 3

**

0

0 5

4

3 2 1 0

Zoysia tenuifolia

4 3 2 1 0

absent present Earthworms

absent present Earthworms

28

Fig. 2 Effects of soil nutrient availability and earthworms on final dry belowground mass (mean + SE) in eight grass species within the low- and high-nutrient quadrants in the heterogeneous soil treatment and within haphazardly chosen quadrants (medium-nutrient treatment) in the homogeneous soil treatment. Letters above bars show which means differed between nutrient treatments within earthworm treatments in cases where nutrient treatments differed (pair-wise comparisons with Bonferroni

0.0

Festuca rubra

1.0 0.8 0.6 0.4 0.2 0.0

1.0 0.8 0.6 0.4

Paspalum notatum b ab a

0.2 0.0

Zoysia matrella

1.0 0.8 0.6 0.4 0.2 0.0

absent present Earthworms

Belowground mass (g/plant)

1.2

0.2

1.2

Belowground mass (g/plant)

Belowground mass (g/plant)

1.2

0.4

low nutrient

1.2

Belowground mass (g/plant)

Belowground mass (g/plant)

1.2

medium nutrient Agrostis stolonifera

0.6

Belowground mass (g/plant)

Belowground mass (g/plant)

0.6

Belowground mass (g/plant)

adjustment based on ANOVA, P = 0.5). See Table 3 for ANOVA.

1.2

high nutrient Elymus dahuricus

1.0 0.8 0.6 0.4 0.2 0.0

Lolium perenne

1.0

b ab

0.8

a

0.6 0.4 0.2 0.0

Poa annua

0.4

0.2

0.0

Zoysia tenuifolia

1.0 0.8 0.6 0.4 0.2 0.0

present absent Earthworms

29

Fig. 3 Relationship between species foraging ability (mean ± SE) and effects on final dry total mass of (A) soil nutrient heterogeneity, (B)

Agrostis stolonifera Elymus dahuricus Festuca rubra Lolium perenne Paspalum notatum Poa annua Zoysia matrella Zoysia tenuifolia

A

1.2

0.9

0.6

0.3

0.0 0.5

0.6

0.7

0.8

Effect of earthworms in homogeneous soil

Foraging ability 1.0

C

0.8

0.6

0.4

0.2

0.0 0.5

0.6

0.7

0.8

Effect of earthworms in heterogeneous soil

Effect of heterogeneity

1.5

Effect of earthworms on effect of heterogeneity

earthworms on effect of heterogeneity, and earthworms in (C) homogeneous and (D) heterogeneous soil. See text for definitions of effects.

B

0.5

0.0

-0.5

-1.0 0.5

0.6

0.7

0.8

Foraging ability 1.0

D

0.8

0.6

0.4

0.2

0.0 0.5

0.6

0.7

Foraging ability

Foraging ability

30

0.8

Supplementary Material

Table S1 ANOVA of effects of grass species, soil heterogeneity, and earthworms on final dry mass and number of tillers of grass. Species is treated as a random factor and other effects as fixed factors. Symbols give P: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01; *** <0.001. Total Effects

Aboveground Belowground

Tiller

df mass

mass

mass

number

Species (S)

7,11

5.82**

5.13**

7.48**

3.01*

Heterogeneity (H)

1,7

20.48**

22.22**

14.49**

41.55***

Earthworms (W)

1,7

18.83**

20.96**

14.18**

14.17**

S×H

7,7

6.70*

5.63*

12.63**

0.81

S×W

7,7

13.75**

10.92**

27.05***

5.06*

H×W

1,7

0.03

0.18

0.83

0.04

7,150

0.29

0.45

0.09

0.57

S×H×W

31

Fig. S1 Effects of soil heterogeneity and earthworms on aboveground mass (mean + SE) of eight grass species. Symbols above pairs of bars show P (linear contrast based on ANOVA) that means for homogeneous and heterogeneous treatments did not differ within earthworm

3

homogeneous Agrostis stolonifera

2

1

*

Aboveground mass (g/plant)

Aboveground mass (g/plant)

treatments: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01.

0

Paspalum notatum

2

* 1

*

0

Zoysia matrella

2

1

0

Aboveground mass (g/plant)

1

Aboveground mass (g/plant)

*

*

1

3

Lolium perenne

* 2

* 1

0 3

Poa annua

2

1

0 3

Aboveground mass (g/plant)

Aboveground mass (g/plant) Aboveground mass (g/plant)

#

3

Aboveground mass (g/plant)

Festuca rubra

2

3

***

2

0

0 3

heterogeneous Elymus dahuricus

3

Zoysia tenuifolia

2

1

0

absent present Earthworms

absent present Earthworms

32

Fig. S2 Effects of soil heterogeneity and earthworms on belowground mass (mean + SE) of eight grass species. Symbols above pairs of bars show P (linear contrast based on ANOVA) that means for homogeneous and heterogeneous treatments did not differ within earthworm

homogeneous Agrostis stolonifera

1.5 1.0 0.5

Belowground mass (g/plant)

2.0

0.0 2.0

Festuca rubra

1.5 1.0 0.5 0.0

*

1.0 0.5 0.0 2.0

Belowground mass (g/plant)

Paspalum natatu

*

#

1.0 0.5

2.0

Lolium perenne

1.5 1.0 0.5

Zoysia matrella

1.5 1.0 0.5 0.0

2.0

Belowground mass (g/plant)

1.5

1.5

0.0

Poa annua

1.5 1.0 0.5 0.0 2.0

Belowground mass (g/plant)

Belowground mass (g/plant)

2.0

heterogeneous Elymus dahuricus

2.0

0.0

Belowground mass (g/plant)

Belowground mass (g/plant)

Belowground mass (g/plant)

treatments: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01.

Zoysia tenuifolia

1.5 1.0 0.5 0.0

absent present Earthworms

absent present Earthworms

33

Fig. S3 Effects of soil heterogeneity and earthworms on number of tillers (mean + SE) of eight grass species. Symbols above pairs of bars show P (linear contrast based on ANOVA) that means for homogeneous and heterogeneous treatments did not differ within earthworm treatments: ns - > 0.1; # - 0.05-0.1; * - 0.01-0.05; ** - 0.001-0.01. 15

homogeneous Agrostis stolonifera

12

Tillers/plants

9 6

0

0

**

9 6

6 3

0

0

Paspalum notatum

12

*

9 6

15

9

0

0 15

Zoysia tenuifolia

12

Tillers/plants

12

#

6 3

Zoysia matrella

Poa annua

12

3

15

#

9

3

15

Lolium perenne

12

Tillers/plants

12

Tillers/plants

15

Festuca rubra

*

6 3

15

Tillers/plants

9

3

Tillers/plants

Tillers/plants

12

Tillers/plants

heterogeneous Elymus dahuricus

15

9 6 3

9 6 3

0

0

absent present Earthworms

absent present Earthworms

34

Highlights 

Little is known about interactive effects of nutrient heterogeneity and earthworms on plant growth.



Both soil heterogeneity and earthworms enhanced plant performance in most species.



Presence of earthworms reduced effects of heterogeneity on growth of some species.



Earthworms in heterogeneous soil may reduce benefits of root foraging for nutrients.

35

36

Research was supported by the National Natural Science Foundation of China (Grants 31570413, 31870610 and 31761123001). The authors declare no conflicts of interest.

37