Drought weakens the positive effects of defoliation on native rhizomatous grasses but enhances the drought‐tolerance traits of native caespitose grasses

Abstract The objective of this study was to evaluate the drought tolerance, compensatory growth, and different plant traits between two native perennial caespitose grasses and two native rhizomatous grasses in response to drought and defoliation. A randomized complete block design at the Swift Current Research and Development Centre (SCRDC) of Agriculture and Agri‐Food Canada (AAFC) examined the effects of water stress and clipping on the plant biomass, plant morphological traits, and relative leaf chlorophyll content (SPAD value) of four native grasses (caespitose grass: Hesperostipa comata and H. curtiseta; rhizomatous grass: Pascopyrum smithii and Elymus lanceolatus). Drought drastically decreased the shoot and root biomass, plant height, number of tillers and leaf growth of P. smithii and E. lanceolatus, as well as the rhizome biomass and R/S ratio of P. smithii. Defoliation had a positive effect on the shoot biomass of P. smithii and E. lanceolatus under well water treatments (100% and 85% of field capacity). However, the compensatory growth of P. smithii and E. lanceolatus significantly declined with increased water stress. In addition, there are no significant changes in plant biomass, plant height, number of tillers and leaves, and SPAD value of H. comata and H. curtiseta under relative dry condition (70% of field capacity). Consequently, these results demonstrated that the rhizomatous grasses possessed a stronger compensation in response to defoliation under wet conditions, but the positive effects of defoliation can be weakened by drought. The caespitose grasses (Hesperostipa species) exhibited a greater drought tolerance than rhizomatous grasses due to the relatively stable plant traits in response to water stress.

Furthermore, the impacts of drought on native grassland ecosystems depend on species composition and interspecific competition of plants to limited resources, because drought tolerance of native plants varies widely from species to species (Shinoda et al., 2010). In addition, the response of plant traits to water stress is crucial to the survival and reproduction of grassland species experiencing drought periods (Tucker, Craine, & Nippert, 2011). Drought can cause a series of reductions in morphologic and physiological functional traits, such as plant height, specific leaf area, leaf water potential, leaf tissue density, and length of roots. Then, these variations of plant traits may lead to a decline in yield and nutritive value (Cenzano, Varela, Bertiller, & Luna, 2013;del Glise et al., 2015;Wellstein et al., 2017). Therefore, an understanding of the quantitative relationship between drought and various plant traits is a key to the utilization of drought-resistant species.
The defoliation of plants by harvest and herbivore grazing is a common phenomenon in the grassland ecosystem. In general, these disturbances could exacerbate the negative effects of environmental stress on plant growth (Bork, Broadbent, & Willms, 2016;Loeser, Sisk, & Crews, 2007). Many studies have shown that resistant plant species have evolved particular adaptive strategies to maintain reproductive capability of plant populations and the stability of the plant community (Norton, Malinowski, & Volaire, 2016;Volaire, 2018;Zwicke, Picon-Cochard, Morvan-Bertrand, Prud'Homme, & Volaire, 2015). For example, resistant plants adopt the tolerance strategy or the avoidance strategy with the various expressions of physiological and morphological traits in response to the combinations of environmental stress and defoliation (Chen, Zhao, Zhang, & Gao, 2013;Feller & Vaseva, 2014;Sonnier, Shipley, & Navas, 2010).
In a semiarid environment, native perennial grasses are often the dominant plants due to superior stress tolerance and competitiveness, and thus preserve the productivity and stability of the plant community (McGlone, Sieg, Kolb, & Nietupsky, 2012;Mischkolz, Schellenberg, & Lamb, 2013;Schellenberg, Biligetu, & Iwaasa, 2012). The dominant species include a variety of grasses with different growth forms (Wallis de Vries, Manibazar, & Gerlham, 1996), such as the perennial caespitose grasses (represented by Stipa or Hesperostipa species) and the perennial rhizomatous grasses (such as Pascopyrum smithii, Elymus lanceolatus, Leymus chinensis, Sorghum halepense, and Cynodon dactylon). In fact, several studies have focused on the response of caespitose and the rhizomatous grasses to various environmental stresses and disturbance in the different grassland types. Chen et al. (2013) noted that Stipa grandis in the steppe of north China increased investment in concentration of defense compounds in leaves as an avoidance strategy to prevent herbivores grazing, and Stipa krylovii utilized the tolerance strategy of rapid growth in response to defoliation under drought stress to get a dominant position. Similar to Stipa species, the perennial rhizomatous grasses possess droughttolerant strategies in plant communities. For example, Pascopyrum smithii can obtain more limited resources by their large deep-root system even though under drought (Dong, Patton, Wang, Nyren, & Peterson, 2014). Additionally, both of the caespitose and rhizomatous grasses have the similar tiller longevity and bud bank densities, but their growth forms differ in number, distribution, and branch of leaves and tillers (N'Guessan, 2007;Ott, 2014).
Moreover, photosynthetic characteristics and leaf traits of caespitose grasses differ from rhizomatous grasses (Lulli et al., 2011;Wang, Zhou, Jiang, Shi, & Xu, 2017). These different plant traits may result in various response mechanisms between the two grass types to defoliation and water stress. Present studies showed that the slower tiller regrowth of Hesperostipa species may be better adapted to drought, and the tiller growth rates responded positively to defoliation (Broadbent, Bork, & Willms, 2017). However, the effects of drought and defoliation on tiller growth rate of rhizomatous grasses are site specific and varied (Bryant, Matthew, & Hodgson, 2015;N'Guessan, 2007). Nevertheless, these works lack quantitative analysis to explore the tipping point of drought tolerance of the caespitose and rhizomatous grasses when coping with water stress, which inhibits the assessments of how drought affects these native plant species.
The Mixed Grassland Ecoregion that forms part of the northern portion of the Great Plains in North America is dominated by native cool-season (C 3 ) grasses (Bailey, Schellenberg, & McCartney, 2010 of 32-42%. The supplemental daylight would be turned on when the natural light energy was less than 500 Wm 2 , and turned off if accumulated light reached 3,620 Wm 2 hr from 7 am to 11 pm every day. The experiment was designed as a randomized complete block with 32 treatments and three replicates, and repeated twice (from 18th March to 5th August 2016 and 5th January to 22nd May 2017).
The 32 treatments consisted of four water treatments with fixed moisture levels: 100% water treatment (100% of field capacity), 85% water treatment (85% of field capacity), 70% water treatment (70% of field capacity), and 55% water treatment (55% of field capacity); two levels of defoliation (No clipping and clipping: all plants were clipped at height of 5 cm); as well as four native grasses: Pascopyrum smithii and Elymus lanceolatus, and two caespitose grasses: Hesperostipa comata and Hesperostipa curtiseta (Figure 1).
The experimental soil was collected at SCRDC, which is an Orthic Brown Chernozem type. Five seedlings of individual species were planted in each pot. Water was applied four times weekly to 100% water treatment, three times weekly to 85% and 70% water treatment, as well as twice weekly to 55% water treatment. We also used the Economy Soil Moisture Tester (Spectrum Technologies, Inc.) as a water dynamic monitor to supplement water. In the clipping treatments, the seedlings were allowed to grow for 12 weeks, and then were clipped twice at 30 days of intervals before the final harvest, and all the clipped plant materials would be added to overall shoot biomass measurements. Co., Ltd., Japan) (Wood, Tracy, Reeves, & Edmisten, 1992). The SPAD value of 10 leaves was measured in each pot. All of these plant trait F I G U R E 1 Four native grasses in this experiment. Upper left: Pascopyrum smithii. Upper right: Elymus lanceolatus. Lower left: Hesperostipa comata. Lower right: Hesperostipa curtiseta indicators were measured for each plant, but we used the mean of the five plants in each pot as a replication. Plant biomass was handharvested, and the plant shoots were dried at 70°C oven for 48 hr, as well as the plant roots were washed and dried for one week before biomass determination. Specifically, the rhizome biomass of P. smithii and E. lanceolatus was separated and weighed. The plant biomass was measured by the accumulation of whole plants per pot, and a pot was regarded as a replication. Additionally, the R/S ratio was determined by root and rhizome biomass (R) divided by shoot biomass (S).

| Statistical analysis
All statistical analyses were conducted using R software (Team, 2016). Two-way ANOVA was used to determine the effects of water, clipping and their interactions on shoot and root biomass, rhizome biomass, R/S ratio, plant height, number of tillers, number of leaves, leaf length, and width, as well as SPAD value within four species, separately. Multiple comparisons were conducted to evaluate the plant biomass, the indicators of plant morphological traits, and SPAD value among the different water levels, and evaluated them between clipping and no clipping treatment at the same water level, separately. The Student's t test was used to detect differences at a significance level of 0.05. Additionally, the relationships among all these plant indicators with the water treatments and clipping treatment were analyzed by correspondence analysis (CA) using R package "ca" (Nenadic & Greenacre, 2011).

| Shoot and root biomass
The effects of water treatment were significant on the shoot and root biomass of P. smithii and E. lanceolatus, as well as the shoot and root biomass of H. curtiseta and H. comata (Table 1). Compared with 100% water treatment, water stress (85%, 70%, and 55% water treatments) gradually decreased the shoot and root biomass of P. smithii (69.4% and 87.6%) and E. lanceolatus (64.6% and 78.1%), as well as the rhizome biomass of P. smithii (74.6%). However, the shoot and root biomass of H. curtiseta and H. comata had a slightly increasing trend under 85% and 70% water treatments, and then significantly decreased under 55% water treatment (Figure 2a,b).
The clipping treatment also had a significant effect on the shoot and root biomass of P. smithii, E. lanceolatus, H. curtiseta, and H. comata (Table 1). However, the effects of interaction of water stress and clipping only were significant on root biomass of P. smithii and E. lanceolatus. After clipping, the shoot biomass of P. smithii and E. lanceolatus was improved 25.6% and 20.3% under 100% water treatment, and increased 15.7% and 37.6% under 85% water treatment. But no significant increase in the shoot biomass of H. curtiseta and H. comata was found for any water treatments ( Figure 2a). In addition, clipping significantly reduced the root biomass of the four plant species and the rhizome biomass of P. smithii under the 100%, 85%, and 70% water treatments (Figure 2b,c).

| R/S ratio
The R/S ratio of P. smithii significantly decreased with the increasing water deficiency under no clipping treatment, which the highest point was 2.98 under 100% water treatment, and the lowest point was 1.28 under 55% water treatment, but the largest R/S ratio appeared at 85% water treatment for E. lanceolatus (2.04), and appeared at 70% water treatment for H. curtiseta and H. comata (1.05 and 1.04).
The clipping treatment resulted in no significant differences of R/S ratio among water treatments for all plant species. Compared with the R/S ratio under no clipping treatment, H. curtiseta and H. comata had a slight reduction under 85% and 70% water treatments, but P. smithii and E. lanceolatus significantly decreased (P. smithii: 72.8% and 71%, E. lanceolatus: 71.2% and 63.8%, respectively) under 100% and 85% water treatments after clipping ( Figure 3).

| Plant morphological traits
For all the plant species, the effects of water stress were significant for the measured plant morphological traits (Table 1)

| Relative leaf chlorophyll content
The relative leaf chlorophyll content (SPAD value) of H. curtiseta and H. comata declined significantly at 55% water treatment. In the no clipping treatment, SPAD value of P. smithii increased with the increasing water stress, but no significant effect of water stress was found (Table 1). However, water stress decreased the SPAD value of P. smithii under clipping treatment. In addition, the effects of clipping on P. smithii, E. lanceolatus, and H. comata were significant (Table 1).
In particular, SPAD values of P. smithii were significantly improved after clipping under all water treatments (Table 2).
TA B L E 1 Results (p-values) of a two-way ANOVA on the effects of water (W) and clipping (CL) treatments, and their interactions on the shoot, root and rhizome biomass, R/S ratio, plant height, number of tillers, number of leaves, leaf length, leaf width, canopy diameter and SPAD value in four native grasses

| Correspondence analysis
Correspondence analysis (CA) of the mixed data for plant traits revealed the different internal relationships among these indicators as well as the correspondence with the water stress and clipping treatments, respectively (Figure 4)

| The rhizomatous grasses response to drought and defoliation
The perennial rhizomatous grasses generally possess superior stem height, leaf area and leaf biomass, and more roots and shoots branched out from nodes of rhizomes in comparison with perennial F I G U R E 2 Boxplots showing shoot biomass (a), root biomass (b) and rhizome biomass (c) of four native grasses under water and clipping treatments with mean (dotted line), median (solid line), quartiles, outliers and the range of data. Letters indicates significant differences (p < 0.05   caespitose grass (Xu & Zhou, 2011). In this study, the shoot and root biomass of P. smithii and E. lanceolatus declined gradually with the exacerbation of water deficiency, which is consistent with previous studies. Eneboe, Sowell, Heitschmidt, Karl, and Haferkamp (2002) noted that drought stress dramatically decreased the growth rate of tillers for P. smithii, and then reduced productivity. Wang and Schellenberg (2012) proposed that the aboveground and belowground biomass of P. smithii had a positive linear dependence, and both of them can be restricted by drought conditions due to its lower photosynthetic capacity and water efficiency in comparison with other grasses. In addition, we detected no significant difference in rhizome biomass of E. lanceolatus among all treatments, but water stress clearly reduced the rhizome biomass of P. smithii, because the strongly creeping rhizomes of P. smithii are more sensitive to drought stress than shoots and roots (Asay & Jensen, 1996;Dong et al., 2014). As expected, P. smithii and E. lanceolatus showed a greater compensation of shoot biomass after defoliation than Hesperostipa species, especially under well-watered conditions, but no compensation of rhizome and root biomass was detected after defoliation.
van Staalduinen and Anten (2005)  Drought has been reported to intensify the responses of plant species to defoliation (Chen et al., 2013;Heitschmidt, Klement, & Haferkamp, 2005). Meanwhile, defoliation also may weaken the negative impact of drought stress through reducing the importance of water availability (Napier, Mordecai, & Heckman, 2016). Our results showed that clipping led to a drastic decline in the R/S ratio of the native grasses, which may be an important emergency mechanism of native plants to damage by allocating the photosynthesis carbon from root system to new shoots and leaves of compensatory growth (Mokany, Raison, & Prokushkin, 2006;Zhao, Chen, & Lin, 2008). However, we did not detect any significant interaction effect of water stress and clipping on the shoot biomass, root biomass of all grasses. This lack of interaction could be caused by the environmental restriction in the greenhouse, for example, experimental pot limited the longitudinal growth of plant roots, and weakened the response of soil-root system to drought and defoliation. Moreover, actual field conditions could be more severe with lesser amounts of soil moisture and more frequent defoliation.
Plant species usually adopt different resistance strategies by distinctive morphological and physiological traits in response to drought and defoliation (Chen et al., 2013;Fornoni, 2011;Volaire, 2008

2) Ac
Notes. Different letters in the table indicate significant difference (p < 0.05). The lower case letters denote the significant differences among the different water treatments under clipping or no clipping treatment in each plant species. The upper case letters denote the significant difference between clipping and no clipping treatment at the same water level.
TA B L E 2 (Continued) species and the rhizomatous grasses. In terms of morphology, leaf traits can be a useful common metric to account for the variation in habitats (Storkey et al., 2013). In P. smithii and E. lanceolatus, leaf length, leaf width, and plant height were observed to have a strong relationship with dry conditions. Water stress limits leaf growth by slowing the rate of cell division and expansion due to loss of turgor, thus reduces plant height (Jaleel et al., 2009;Poormohammad Kiani et al., 2007), and this is probably the main reason that the rhizomatous grasses decreased their photosynthetic activity and thus plant biomass under drought stress. Compared to other plant trait parameters measured, the number of rhizomatous tillers and shoot biomass was more closely linked with clipping treatment under relatively sufficient water conditions. On the one hand, the stable number of tillers is an important tolerance mechanism for maintaining the basic productivity under the drought stress (Busso & Richards, 1995;Zhang & James, 1995); on the other hand, the rhizomatous grasses may inhibit the increasing tillers to reallocate resources to contribute to compensatory growth of leaves (Broadbent et al., 2017;van Staalduinen & Anten, 2005;Zhao et al., 2008). In addition, we found that the number of leaves, root biomass, and rhizome biomass in two rhizomatous grasses correlated with the 100% and 85% water treatments under no clipping treatment, even though the  (Shipley & Meziane, 2002). For the plant physiological trait, we used relative leaf chlorophyll content (SPAD value) as a proxy for leaf photosynthetic capacity (Croft et al., 2017). We found positive effects of clipping on SPAD value in these native grasses, which is an important mechanism of leaf regrowth for defoliation tolerance (Briske & Richards, 1995;N'Guessan, 2007). Moreover, the SPAD value of P. smithii and E. lanceolatus after clipping showed an decreasing trend with water stress, because clipping can remove those old and dead tissues that have the lower leaf chlorophyll content, and then the negative effect of drought stress on leaf chlorophyll content was highlighted with the remnant leaves regrowth (Zhao et al., 2008). Therefore, our results suggest that drought not only can decrease shoot and root biomass, tiller and leave growth of P. smithii and E. lanceolatus, but also worsen the defoliation effects on plant traits and grass yield of the two rhizomatous grasses. rolling mechanism to decrease transpiration, reduce water consumption by lower productivity, and improves leaf N concentration to enhance photosynthetic rate, as well as utilize the poikilohydric-type habits in response to drought stress (Balaguer et al., 2002;Shi et al., 2015). In this study, we detected that the shoot and root biomass of Hesperostipa species had a slight reduction under wet conditions, which indicated excessive moisture may weaken the plant growth by limiting photosynthetic activity (Xu & Zhou, 2011).
The SPAD value of two caespitose grasses, a physiological trait, exhibited a stronger positive connection to the clipping treatment with 100% and 85% water treatment than the rhizomatous grasses.
These results indicate that the regenerated leaves of Hesperostipa species after defoliation may improve leaf chlorophyll content and photosynthetic capacity under sufficient soil moisture (Balaguer et al., 2002). However, it is worth mentioning that a few differences were detected in our results between two Hesperostipa species.

| CON CLUS ION
In this study, we found that the two native rhizomatous grasses defoliation under wet and dry conditions. These results demonstrated that drought is a key factor to inhibit the compensation of rhizomatous grasses after defoliation. The Hesperostipa species is considered to be the superior for adaptation to drought in comparison to the native rhizomatous grasses.

ACK N OWLED G M ENTS
This experiment was supported financially by AAFC Growing Forward 2 and the Beef Science Cluster. Thanks to L. Fast, S. Lim, S. Bender, E. Stuart, I. Piche, P. Coward for their assistance in this greenhouse study.

CO N FLI C T O F I NTE R E S T
None declared.