https://cpw.cvlcollections.org/files/original/479dafc1c693daabfbda4165ae02cb68.pdf 455c07e605bb56dba8d3c3ed7d3f7865 PDF Text Text The research in this publication was partially or fully funded by Colorado Parks and Wildlife. Dan Prenzlow, Director, Colorado Parks and Wildlife • Parks and Wildlife Commission: Marvin McDaniel, Chair • Carrie Besnette Hauser, Vice-Chair Marie Haskett, Secretary • Taishya Adams • Betsy Blecha • Charles Garcia • Dallas May • Duke Phillips, IV • Luke B. Schafer • James Jay Tutchton • Eden Vardy �UN DECADE ON ECOSYSTEM RESTORATION R E S E A R C H A RT I C L E Cultivars of popular restoration grass developed for drought do not have higher drought resistance and do not differ in drought-related traits from other accessions Magda Garbowski1,2,3 , Danielle B. Johnston4 , Cynthia S. Brown1,2 Numerous functional traits have been identified as key contributors to plant performance under drought. However, many of these traits, specifically root traits, are rarely considered in the development of native plant cultivars. In this study, we assessed whether cultivars of the perennial grass Elymus trachycaulus (Slender wheatgrass) developed for drought differ in (a) drought resistance (i. e. a plant’s ability to maintain aboveground biomass productivity under water deficit), (b) aboveground and belowground traits, and (c) trait responses to drought from other accessions (i.e. other cultivars, wild accessions). We also evaluated trait plasticity, assessed whether multivariate trait relationships varied between control and drought conditions, and determined which suites of traits are related to drought resistance. We worked with seedlings at two developmental stages to assess whether patterns vary ontogenetically. E. trachycaulus cultivars developed for drought did not differ from other accessions in drought resistance or traits related to drought-coping strategies. The effects of drought and accession on drought resistance, traits, and trait plasticity varied by developmental stage, but relationships among traits varied little between the two developmental stages. A primary axis of functional variation related to resource acquisition (plant height, root length, root tips) was consistently associated with drought resistance. However, which secondary axes were related to drought resistance varied by developmental stage and moisture condition. Our results suggest that traits and performance of commonly used cultivars ought to be reexamined to determine whether they are actually the best candidates for revegetation projects in specific contexts. Key words: cultivars, drought strategies, functional traits, intraspecific trait variation, ontogeny, root traits Implications for Practice • • • • Traits that are traditionally selected for in cultivar development may not be advantageous in contemporary restoration settings in which plants must cope with novel environmental stressors. Determining which traits or suites of traits improve plant performance in specific contexts (e.g. in water- limited systems) is an important first step in identifying new sources of native plant materials for revegetation in changing landscapes. As “drought cultivars” of Elymus trachycaulus did not produce more biomass than other cultivars or wild accessions under drought conditions in our study, identifying new sources of this and other species for revegetation of drought-prone systems may be warranted. Researchers and native plant producers ought to reexamine whether cultivars developed for specific purposes are actually the best candidates for revegetation projects. Introduction Natural and anthropogenic disturbances have resulted in degradation across approximately 70% of the world’s terrestrial July 2021 Restoration Ecology Vol. 29, No. 5, e13415 ecosystems (IPBES 2019). In an attempt to mitigate some of the damage, the science and practice of restoration ecology has grown rapidly over the last several decades. Restoration of degraded ecosystems is now considered a key tool for curbing biodiversity loss, with economic and ecological returns that far exceed monetary inputs (Menz et al. 2013). Effective and economical revegetation practices will be central to meeting restoration goals outlined in the United Nations’ declaration of 2021– 2030 as the “Decade on Ecosystem Restoration”. However, successful restoration of terrestrial ecosystems often hinges on the Author contributions: MG, DBJ, CSB conceived of this research; MG completed data collection, analyses, and writing of the manuscript; MG, DBJ, CSB edited the manuscript. 1 Graduate Degree Program in Ecology, Colorado State University, 102 Johnson Hall, Fort Collins, CO, 80523, U.S.A. 2 Department of Agricultural Biology, Colorado State University, 307 University Ave., Fort Collins, CO, 80521, U.S.A. 3 Address correspondence to Magda Garbowski email magda. garbowski@colostate.edu 4 Colorado Division of Parks and Wildlife, 711 Independent Ave., Grand Junction, CO, 81505, U.S.A. © 2021 Society for Ecological Restoration. doi: 10.1111/rec.13415 Supporting information at: http://onlinelibrary.wiley.com/doi/10.1111/rec.13415/suppinfo 1 of 12 �Drought cultivars underperform in drought settings availability of high-quality seeds of native species able to establish and persist in changing ecosystems (Broadhurst et al. 2015). Because seeds of many species are often not commercially available (Ladouceur et al. 2017) or are cost-prohibitive to use (Chivers et al. 2016), restoration practitioners often use widely available cultivars for revegetation projects. A cultivar is a “distinct, intentionally developed subset of a species that will behave uniformly and predictably when grown in an environment to which it is adapted” (Aubry et al. 2005). Despite their abundant use, literature documenting cultivar selection and development is difficult to find. From the literature that is available, it appears that cultivars are often selected for characteristics such as vigor, height, and yield. In addition, selection trials are often conducted far from where seeds are ultimately used, in monoculture stands, and sometimes under irrigated conditions. This selection and development process may lead to the release and use of cultivars with traits that are not advantageous in realistic restoration settings (Leger & Baughman 2015). However, recent research has identified numerous traits which are not commonly selected for in cultivar development as key contributors to plant success in restoration (e.g. Larson & Funk 2016; Larson et al. 2020; Leger et al. 2020). A growing number of restoration researchers are adopting trait-based approaches to identify traits associated with plant performance in restoration settings. With these approaches researchers aim to discern and ultimately predict patterns of plant establishment, performance, and survival based on standardized and quantifiable plant characteristics (i.e. traits) rather than species identities (Violle et al. 2007). With information about how traits influence various performance measures such as biomass production or survival, researchers and practitioners can begin to unravel the underlying mechanisms driving community assembly in restored systems (e.g. Funk et al. 2008; Laughlin 2014). Various and sometimes surprising traits have been related to plant performance in restoration settings using trait-based methods. For example, working with the perennial grass Elymus elymoides, Kulpa and Leger (2013) found that small plants with small seeds and early phenology had high rates of survival in restored areas of the Great Basin and Foxx and Kramer (2020) found E. elymoides individuals with high root tip numbers to be effective competitors with the invasive grass Bromus tectorum. Similarly, Leger and Baughman (2015) identified early phenology, small plant size, and high root allocation to be strong predictors of plant performance across 420 seeding treatments implemented throughout the western United States (US hereafter). In all of these studies, traits which were found to improve plant performance were not those that are traditionally selected for during cultivar development. Drought tolerance or drought resistance are mentioned in nearly a third of published materials on cultivars used in restoration throughout the western United States (Leger & Baughman 2015). This is unsurprising as drought hinders the recruitment of establishing plants in restored dryland systems (e.g. Booth et al. 2003; Hardegree et al. 2012; Garbowski et al. 2020a, 2021a). However, information about how drought tolerance or resistance are assessed or measured in cultivar development trails is rarely included in release documents or plant 2 of 12 brochures. According to a recently refined framework that characterizes plant strategies for coping with drought in relation to traits (Volaire 2018), drought-tolerant plants are able to withstand low tissue hydration and ultimately survive drought. Drought-resistant plants, on the other hand, employ dehydration escape or avoidance strategies which allow them “to maintain satisfactory growth and production under moderate water deficit” (May & Milthorpe 1962). Determining whether cultivars traditionally bred for drought actually possess traits related to specific drought-coping strategies would clarify whether they are the most appropriate candidates for restoration in drought-prone systems. Root traits may be particularly informative for understanding plant establishment, survival, and performance in drought settings (reviewed in Garbowski et al. 2020b). Yet, root traits are rarely considered in the development of native plant materials. Root traits associated with resources conservation such as high root tissue density (RTD) may be particularly useful for identifying drought-tolerant populations or species. For example, populations of the perennial grass Dactylis glomerara with high RTD were found to have high survival after seasonal drought (Bristiel et al. 2018). Similarly, Hanslin et al. (2019) found high RTD to be part of a general conservative strategy various grass species used to cope with water stress. In contrast, root traits associated with rapid resource acquisition, such as high total root length and high specific root length (SRL), may be particularly important for drought resistance. Such acquisitive traits may allow plants to optimize water acquisition and maintain aboveground growth (e.g. Comas et al. 2014; Volaire 2018). As the success of restoration projects often depends on the plants maintaining biomass production under stressful conditions, identifying which traits or suites of traits underpin drought resistance in plant materials may provide new insight about the mechanisms driving revegetation outcomes. Plant strategies for growth and survival are often comprised of suites of traits which together affect performance (e.g. Garnier et al. 2016). Therefore, identifying coordinated axes of functional variation can provide a more comprehensive understanding of how plants respond to and cope with environmental stressors such as drought (Volaire 2018). However, because growing conditions (e.g. Poorter et al. 2016) and plant age (e.g. Garbowski et al. 2021b) affect trait values, making trait-based comparisons among species or populations across different environments or developmental stages is challenging. Assessing the relative plasticity of traits in response to different resource levels allows researchers to identify plastic traits which should be studied with caution versus stable traits which may be more reliable for comparison across diverse systems and developmental stages (e.g. Kramer-Walter & Laughlin 2017; Garbowski et al. 2021b). Furthermore, as plants may adjust multiple traits to cope with resource stress simultaneously (Craine et al. 2001), relationships among traits may vary depending on environmental condition. Identifying stable traits and trait relationships that enhance seedling performance under a variety of moisture conditions is an essential step in applying trait-based approaches to dryland revegetation. Elymus trachycaulus (Slender wheatgrass, E. trachycaulus hereafter) is an abundant, short-lived perennial grass with a Restoration Ecology July 2021 �Drought cultivars underperform in drought settings distributional range that extends from Alaska to eastern Canada and south to Mexico (Hitchcock 1951). It is readily grazed by wildlife and provides food and cover for small mammals and bird species (Tilley et al. 2011). It is widely used in restoration in the western United States, and several cultivars of E. trachycaulus have been developed for specific restoration needs including performance under drought. However, because information on development trials of E. trachycaulus is difficult to find, little is known about whether “drought cultivars” (i.e. “Pryor” and “First Strike”) outperform other groups in moisture deficit conditions or have superior traits from other accessions related to drought-coping strategies. We worked with several cultivars and wild accessions (i.e. collected from noncultivated naturally occurring populations) of E. trachycaulus to evaluate drought resistance and assess differences among the groups in leaf and root traits that prior research has identified as important contributors to drought-coping strategies. In addition, we assessed trait correlations in control and drought conditions and identified which suites of traits are associated with drought resistance. We worked with seedlings at two early developmental stages—10days-old and 24 -days-old—because prior research has identified this period as critical to seedling establishment (e.g. Rowe & Leger 2010; Larson et al. 2016) and one during which traits can vary substantially (Garbowski et al. 2021b). With this study we sought to answer the following research questions: (1) Do cultivars developed for drought differ from other accessions (i.e. cultivars developed for other purposes or wild accessions) in drought resistance, traits, or responses of traits to drought? (2) Do relationships among traits vary between control and drought conditions and which suites of traits are correlated with aboveground biomass production in each case? (3) Which traits are least plastic (i.e. stable) between control and drought conditions and therefore reliable candidates for future study? (4) Do patterns observed in Questions 1–3 vary during early plant development (i.e. with ontogeny)? Methods Plant Materials Selection We worked with seven accessions of Elymus trachycaulus: two cultivars developed for drought (“Pryor” and “First Strike”), two other cultivars (“San Luis” and “Revenue”), and three wild accessions from areas that differ in annual precipitation amount (“NM Dry”, “CO Mesic”, “UT Wet”). Cultivars were purchased from Granite Seed Company in Denver, Colorado, and seeds of wild accessions “NM Dry” and “UT Wet” were obtained from the United States Department of Agriculture Germplasm Research Information Network (USDA GRIN). Seeds of the “CO Mesic” accession were obtained from the Upper Colorado Plant Materials Center in Meeker, Colorado. All seeds used in the experiment were harvested from plants grown for one generation in a common greenhouse environment. July 2021 Restoration Ecology Details about the cultivar development process were gathered from a variety of sources including release notes, planting guides, and articles of registration in peer-reviewed journals. The “Pryor” cultivar originates from a saline upland site in Carbon County, Montana, was “tested for more than 9 years against improved cultivars and native collections,” has consistently been “rated superior in seedling vigor, salt, and drought tolerance, forage and seed production, and stand longevity” and “it is capable of withstanding periodic flooding and extended drought” (Release Brochure for Pryor Slender wheatgrass [Elymus trachycaulus], 2012). The “First Strike” cultivar is a composite of three collections from Colorado and Wyoming, was released in 2006, and according to Jensen et al. (2007) was chosen for “persistence and overall plant vigor in response to drought.” The “San Luis” cultivar was developed from seeds collected near the San Luis Valley of Colorado in 1975 and was selected for “outstanding establishment and longevity of stand characteristics” (USDA Release Note). The “Revenue” cultivar originated from Saskatchewan, Canada, and was selected from over 75 collections evaluated between 1959 and 1970 for “rapid establishment, forage and seed yield, and salinity tolerance” (Tilley et al. 2011). We selected three wild accessions of E. trachycaulus to include in our study from areas that span a range of precipitation conditions. The “NM Dry” (USDA GRIN accession: LLPMC5) accession was collected from central New Mexico from an area that receives only 260 mm of rainfall annually and the “CO Mesic” accession was collected in central Colorado from an area that receives 425 mm of precipitation annually. On the wettest end of the range was “UT Wet” (USDA GRIN accession: UT933-383) from southwestern Utah, collected in an area that receives 750 mm of precipitation annually. Details about accessions are provided in Table S1. Precipitation data for each wild accession collection site were obtained from 30-year averages available through the National Oceanic and Atmospheric Administration (NOAA 2019). Plant Growth To allow adequate time for harvesting and trait measurements, seeding was staggered over 1 month, between 15 May and 21 June, 2019. Seeds of each accession were randomly selected every other day and planted into containers (Deepots D60, Stuewe & Sons, Inc. D20T) filled with porous ceramic media (Profile Greens Grade). This media is suitable for growing a variety of plants as it has a high water and nutrient-holding capacity, good drainage characteristics, and its coarse texture allows roots to be easily extracted for analysis. Prior to seeding, approximately 20 g of field soil collected in southwestern Colorado, a location distinct from all collection sites, was added to the top 5 cm of each pot. Immediately after seeding, pots were mist watered every 30 minutes for 1 minute until seedlings emerged. After emergence, plants were moved to ambient greenhouse conditions: 18� C for 12 hours during the day and 13� C at night with alternating 12-hour light versus dark. Upon emergence, seedlings were randomly assigned to one of two watering treatments (control: 40 mL twice a day or drought: 3 of 12 �Drought cultivars underperform in drought settings 10 mL twice a day) and developmental stage (10-days-old or 24-days-old). These watering treatments translate to roughly 25% and 12% volumetric water content in our growing medium. We randomized individual plants on the same greenhouse bench every 3 days throughout the duration of the experiment. Every third day, we watered plants with a fertilizer solution (Cal– Mag Special; 15-5-15). On average seven plants were grown and measured for each accession × treatment × harvest combination (seven accessions × 2 treatments × 2 harvests × 7 replicates = 196 plants total). Trait Measurements We measured the following traits from each plant at the 10- and 24-day developmental stage: height, specific leaf area (SLA), specific root length (SRL), root tissue density (RTD), number of root tips, average root diameter, and total root length according to standard protocols (Perez-Harguindeguy et al. 2013; Freschet et al. 2020). In addition, at each harvest, we measured above- and belowground biomass and calculated the root mass ratio (RMR) of each plant. Upon harvesting, we scanned fresh leaves and roots and used WinRHIZO software with a resolution set to 600 dpi to obtain measures of leaf and root area and root diameter, length, and volume. These values were used to calculate SLA, SRL, and RTD. Analyses We completed all analyses in R (R Core Team, 2020). To examine how water treatment and accession affected multivariate trait space (height, SLA, SRL, RTD, root tips, root diameter, root length, RMR) at each developmental stage, we used perMANOVA (permutation-based multivariate analysis of variance), with 999 permutations and Euclidean distances (vegan package in R; Oksanen & Blanchet, 2016). We used linear models to analyze the effects of accession and drought treatment on aboveground, belowground, and total plant biomass as well as individual traits. Trait values were transformed to ensure homogeneity of variance and normally distributed residuals. We applied a log transformation to SLA, SRL, and a square root transformation to RTD, root diameter, root length, aboveground biomass (24-day developmental stage only), and root biomass (24-day developmental stage only). To determine whether trade-offs among traits were consistent across treatments, we qualitatively interpreted results of principal components analyses (PCA) (psych package in R; Revelle 2021). We determined which traits were correlated with performance by regressing aboveground plant biomass against PCA axes in multiple regression models. We calculated plasticity for each trait between control and drought conditions in accordance with recommendations by Valladares et al. (2006) where PI (plasticity index) = (maximum mean – minimum mean)/maximum mean. Plasticity values were determined at the accession level. This allowed for accession to be used as the unit of replication in one-way ANOVA models with the absolute value of the PI for each trait included as the response and trait included as the predictor. In this way, we were able to assess which traits were most versus least plastic between the two moisture conditions. 4 of 12 Results Multivariate trait space was affected by accession and drought treatment at both the 10-day (Accession p = 0.02, R2 = 0.14, F = 2.08; drought p = 0.01, R2 = 0.07, F = 6.77) and 24-day harvest (Accession p = 0.001, R2 = 0.14, F = 3.04; drought p = 0.001, R2 = 0.27, F = 35.26). Results for drought resistance (Table S2; Fig. 1), trait differences (Table S2; Figs. 2 & S1), and trait plasticity (Figs. 4 & S3) varied between the 10-day and 24-day developmental stage. Overall, fewer differences were detected at the 10-day developmental stage than at the 24-day developmental stage. Few differences between the two developmental stages were observed in multivariate trait relationships (Table 1; Figs. 3 & S3). Performance At the 10-day developmental stage, drought reduced total (p = 0.002; F = 10.33), aboveground (p = 0.009; F = 7.21), and belowground (p = 0.002; F = 10.53) biomass across all accessions (Table S2; Fig. 1). Across both control and drought treatment, wild accession “NM Dry” had greater aboveground biomass than wild accession “UT Wet” (Fig. 1). At the 24-day developmental stage, drought by accession interactions were observed for total (p = 0.018; F = 2.72), aboveground (p = 0.031; F = 2.44), and belowground (p = 0.042; F = 2.29) biomass (Table S2; Fig. 1). However, few differences among accessions were observed within control treatment, and none were observed within drought treatment. In control treatment, total, aboveground, and belowground biomass values of wild accession “NM Dry” were greater than values for “First Strike” (Fig. 1). Within accessions, differences between control and drought treatments in biomass measures were only observed in wild accessions “NM Dry” (lower total, aboveground, and belowground biomass with drought) and “UT Wet” (lower total and aboveground biomass with drought) (Fig. 1). Individual Traits At the 10-day harvest, accession by drought interactions were not detected for any traits besides RMR (p = 0.048; F = 2.21; Table S2; Figs. S1A–H). At this developmental stage, RMR of accession “UT Wet” under drought was higher than RMR of “Pryor” under drought and accession “NM Dry” under drought and control conditions (Fig. S1H). Across all accessions, drought treatment resulted in reduced height (Fig. S1B), SRL (Fig. S1C), root length (Fig. S1D), and number of root tips (Fig. S1F). Accession effects were observed for height (Fig. S1B), root length (Fig. S1D), and root diameter (Fig. S1G), such that the “First Strike” cultivar had lower height and root diameter than all other accessions and lower root length than all other cultivars. No accession or drought effects were observed for SLA (Fig. S1A) or RTD (Fig. S1E) at the 10-day developmental stage. At the 24-day developmental stage, interactions between accession and drought treatment were observed for height (Fig. 2B), SRL (Fig. 2C), root length (Fig. 2D), and number of root tips (Fig. 2F) (Table S2). Despite these interactions, specific Restoration Ecology July 2021 �Drought cultivars underperform in drought settings 10 days 5.0 Total biomass: Drought effect: p < 0.01, F = 10.33 Aboveground biomass: Drought effect: p < 0.01, F = 7.21 Belowground biomass: Drought effect: p < 0.01, F = 10.53 ab ab ab ab a ab b Biomass (mg) 2.5 Treatment Control Drought 0.0 2.5 (A) 24 days A a 20 Biomass (mg) ABC abc 10 AB ab ABC abc ABC abc ABC abc BC bc BC c BC c C c BC c BC c C c Treatment C c Control Drought 0 c b b ab c ab ab ab abc c ab ab (B) Wild Accession UT Wet Wild Accession CO Mesic Wild Accession NM Dry Cultivar San Luis Cultivar Revenue Drought Cultivar Pryor a Drought Cultivar First Strike 10 b Figure 1. Means for aboveground (above y = 0, differences indicated with lowercase letters), belowground (below y = 0, differences indicated with lowercase letters), and total biomass (bars above and below y = 0, differences indicated with uppercase letters) for E. trachycaulus accessions under control (gray bars) and drought (red bars) conditions at the (A) 10-day and (B) 24-day harvest. Bars represent � SE. In (A), lowercase letters above bars indicate significant differences at the p < 0.05 level among accessions in aboveground biomass, averaged over treatments. In (B), lowercase letters above and below bars indicate significant differences at the p < 0.05 level among accessions and treatments in above- and belowground biomass, respectively. Uppercase letters above bars indicate significant differences at the p < 0.05 level among accessions and treatments in total biomass. differences in traits among accessions within treatments were limited. Within control treatment, wild accession “NM Dry” obtained greater height than all other accessions (Fig. 2B) and had greater root length than the “First Strike” cultivar (Fig. 2A). No significant differences in traits among accessions were observed within drought treatment. Across all accessions, July 2021 Restoration Ecology drought treatment resulted in lower SLA (Fig. 2A), higher RTD (Fig. 2E), and higher root diameter (Fig. 2G). An accession effect was observed for root diameter such that wild accession “NM Dry” had a higher diameter than all other accessions besides the “San Luis” cultivar which had a higher root diameter than accession “UT Wet” (Fig. 1G). An accession effect was 5 of 12 �Drought cultivars underperform in drought settings SLA (B) 40 Height [cm] 30 20 10 (C) SRL [m g−1] ab 10 d bcde abcde e abcde de cde bcd bcd cd cd cd cd (D) abcde abcde abcd bcd bcd bcd bc bcd 5 a abcde b 15 SRL abc a 20 Drought effect: p < 0.01, F = 10.28 300 200 Height 25 cde 100 Length [cm] SLA [mm2 mg−1] (A) Root Length 200 150 100 abc abc bc ab ab a abc abc bc bc c abc abc bc 50 0 Root Tips a 400 0.15 0.10 200 abc abc Drought effect: p < 0.001, F = 14.89 bc (H) bc C AB AB AB A B AB B Wild Accession UT Wet BC Wild Accession CO Mesic A Wild Accession NM Dry AB abc Cultivar San Luis BC c bc abc Root Mass Ratio Drought Cultivar Pryor BC abc abc 0 Root Diameter BC a abc Drought Cultivar First Strike 0.6 RMR 0.35 0.30 0.25 0.2 Wild Accession UT Wet Wild Accession CO Mesic Wild Accession NM Dry Cultivar San Luis Cultivar Revenue Drought Cultivar Pryor Drought effect: p < 0.001, F = 12.62 Drought Cultivar First Strike 0.4 Accession Cultivar Revenue 0.05 300 ab 100 (G) Diameter [mm] (F) RTD Tips RTD [mg mm−3] (E) Treatment First Strike Revenue NM Dry Pryor San Luis CO Mesic UT Wet Control Drought Figure 2. Trait means for each accession used in the study under control (circles) and drought (square) conditions at the 24-day harvest. Orange symbols show “drought cultivars,” blue symbols show “other cultivars,” and gray symbols show “wild accessions”. Bars represent � SE from the mean. Lowercase letters indicate significant differences at the p < 0.05 level among accessions and treatments. Uppercase letters indicate significant differences among accessions averaged across control and drought treatments. Model statistics for drought effects are provided. SLA, Specific leaf area; SRL, Specific root length; RTD, Root tissue density. 6 of 12 Restoration Ecology July 2021 �Drought cultivars underperform in drought settings Table 1. Results of principal components analyses of the trait correlation matrices at the 10-day and 24-day harvests under control and drought conditions. Eigenvectors >j0.6j are in bold. Total variance explained by each rotated component (RC) is provided in the Var.explained row of the table. Control: 10 day SLA HT SRL Length RTD Tips Diameter RMR Var.explained Drought: 10 day RC1 RC2 RC3 RC1 RC2 RC3 0.15 0.88 0.00 0.93 0.06 0.88 0.42 0.39 35% −0.11 0.01 −0.88 0.09 0.91 0.08 0.05 0.58 24% 0.68 −0.12 0.28 0.07 0.05 0.14 −0.62 0.56 16% 0.03 0.92 −0.07 0.94 0.16 0.90 0.41 0.02 33% −0.18 −0.05 −0.68 0.17 0.90 0.12 −0.35 0.74 26% 0.83 −0.21 0.51 −0.04 −0.11 0.03 −0.65 0.12 18% Control: 24 day SLA HT SRL Length RTD Tips Diameter RMR Var. explained Drought: 24 day RC1 RC2 RC3 RC1 RC2 RC3 0.08 0.88 −0.15 0.91 −0.30 0.87 0.46 −0.09 34% 0.35 −0.10 0.85 0.08 −0.91 0.11 0.32 0.15 23% 0.73 0.11 0.14 −0.01 0.09 0.17 −0.33 −0.81 17% 0.45 0.69 0.09 0.92 −0.20 0.90 0.02 0.22 30% 0.07 −0.09 −0.86 −0.05 0.88 −0.01 −0.04 0.85 28% 0.18 0.34 −0.46 −0.20 −0.23 −0.20 0.94 −0.06 17% Control Drought 3 (A) 3 (B) 2 2 SRL RC2 − 28% variance explained RC2 − 23% variance explained RTD 1 SLA MR RMR 0 Diameter Tips LTips Length Length HT −1 RTD −2 RMR Accession Drought Cultivar First Strike Drought Cultivar Pryor 1 Cultivar Revenue SLA S 0 Diameter m t HT Cultivar San Luis Tips Length Wild Accession NM Dry −1 Wild Accession CO Mesic Wild Accession UT Wet −2 SRL −2 −1 0 1 2 RC1 − 34% variance explained 3 −2 −1 0 1 2 RC1 − 30% variance explained 3 Figure 3. The first two varimax-rotated principal components (RC) plotted for traits measured at the 24-day harvests under (A) control and (B) drought conditions. Orange symbols show “drought cultivars”, blue symbols show “other cultivars” and gray symbols show “wild accessions.” Points show means and bars show SE for each accession. HT, height; RMR, root mass ratio; RTD, root tissue density; SLA, specific leaf area; SRL, specific root length; tips: root tips. also observed for RMR such that across both control and drought treatments, the “San Luis” cultivar had a higher RMR than wild accessions “NM Dry” and “UT Wet” (Fig. 2H). July 2021 Restoration Ecology Relationships Among Traits, and Trait Plasticity Relationships among traits varied little between the 10-day and 24-day harvests and between control and drought conditions 7 of 12 �Drought cultivars underperform in drought settings Discussion Drought Resistance Figure 4. Boxplots show median, first, and third quartile, and 95% confidence intervals of plasticity indices for each trait at the 24-day harvest. Plasticity index = j (maximum mean – minimum mean)/maximum mean j. Different letters denote significant differences in plasticity in traits at the p < 0.05 level. Trait abbreviations are as follow: SLA, specific leaf area; SRL, specific root length; RTD: root tissue density; length: total root length; RMR: root mass ratio; tips: number of root tips. (Table 1; Figs. 3 & S2). Traits related to aboveground (i.e. height) and belowground resources acquisition (i.e. root length, number of root tips) consistently loaded onto RC1 and explained between 30% and 35% of variation within each developmental stage and treatment combination. Traits related to root tissue construction (i.e. SRL and RTD) consistently loaded onto RC2 and explained between 23% and 28% of the variation in traits. Under drought treatment, RMR also loaded onto RC2. At the 10-day developmental stage, SLA and root diameter loaded onto RC3. At the 24-day harvest SLA and RMR loaded onto RC3 under control conditions, and only root diameter loaded onto RC3 under drought conditions. The third RC explained between 16% and 18% of the variation in traits. RC1 (height, root length, number of root tips) was positively related to aboveground biomass production at both developmental stages under both control and drought conditions. At the 10-day developmental stage, RC3 (SLA, root diameter) was also retained in models and together these RCs explained 63% and 49% of the variation in aboveground biomass under control and drought conditions, respectively. Despite differences in which traits loaded onto axes, all RC axes were related to aboveground biomass at the 24-day developmental stage in control and drought conditions. The resulting models explained 87% and 75% of the variation in aboveground biomass production in control and drought conditions, respectively. No differences in plasticity of traits under control versus drought conditions were observed at the 10-day developmental stage (Fig. S3). At the 24-day developmental stage, average root diameter had the lowest plasticity value of all traits and had a significantly lower plasticity index than height, RTD, root length, RMR, and number of root tips (Fig. 4). 8 of 12 Unlike in other studies which have found cultivars to have superior performance under drought conditions (e.g. Zwicke et al. 2015), we observed few differences in total, aboveground, and belowground biomass between drought cultivars and other accessions. By choosing biomass production under moisture deficit as our measure of performance we assessed the drought resistance of accessions, or “the ability for plants to maintain growth and production under moderate water deficit” (May & Milthorpe 1962). The ability of plants to continue growing under moderate moisture deficit is undoubtably important in restored systems as biomass production is foundational to many restoration goals such as improving forage for livestock, maintaining ecosystem productivity, and resisting invasion by nonnative species. However, drought resistance is only one measure of performance and several studies focused on aboveground (e.g. Volaire & Barkaoui 2013; Norton et al. 2014), belowground (e.g. Bristiel et al. 2018), and whole-plant traits (e.g. Zwicke et al. 2015; Balachowski & Volaire 2018) of perennial grass species have identified important trade-offs between drought resistance and other performance measures such as survival under severe drought. Although we observed no differences in drought resistance among accessions, it is possible that accessions may differ in other, unmeasured performance metrics such as survival under severe drought. It is also possible that traits may differ between plants grown in greenhouse versus field settings (Poorter et al. 2016). However, recent research has found that traits obtained from greenhouse-grown plants are predictive of plant success in field settings (Leger et al. 2020). Identifying and considering specific traits in relation to both drought resistance and drought tolerance (e.g. Volaire & Barkaoui 2013; Volaire 2018) in natural settings will be an imperative next step for selecting appropriate plant materials and for meeting site-specific restoration goals. Individual Traits As in other studies assessing intraspecific variability of traits related to drought in perennial grass species (e.g. Lelièvre et al. 2010; Balachowski & Volaire 2018; Bristiel et al. 2018), we found several differences in traits among accessions of E. trachycaulus. However, cultivars developed for drought did not clearly differ in traits related to drought-coping strategies from other accessions. Rather, wild accessions appeared to differ from one another as well as cultivars in several traits. Furthermore, when differences in traits were observed, they were observed primarily in control not drought conditions. The limited accession by drought treatment interactions we observed at the 10-day developmental stage may result from the strong influence of seed provisioning on plant performance during the first days of seedling growth (e.g. Leck et al. 2008; Larson et al. 2020) or a delay in morphological plant responses to water stress (Sun et al. 2020). RMR was the only trait for which an accession by drought interaction was observed at the 10-day developmental stage. As shifts in allocation are highly Restoration Ecology July 2021 �Drought cultivars underperform in drought settings responsive to resource limitation (e.g. Freschet et al. 2015), they may also be among the first adjustments growing seedlings make when they encounter changing environmental conditions. With lower plant height, root length, and root diameter, only one cultivar, “First Strike,” differed from other accessions at the 10-day developmental stage. At the 24-day developmental stage, accession differences were observed in several key traits associated with plant strategies for coping with drought. Notably, wild accession “NM Dry” appeared to possess traits associated with drought avoidance or escape, both strategies that enhance drought resistance. As in another recent study focused on perennial grasses from the western United States (Hoffman et al. 2020), we found that this accession, which originates from an area receiving just 260 cm of precipitation annually, had traits associated with rapid resource acquisition and growth, key characteristics of drought avoidance. The “NM Dry” accession had high root length, a trait that prior research has identified as important for rapid and efficient water acquisition (Comas et al. 2014). This accession also had higher root diameter than most of the other accessions. This result aligns with a recent study that identified root diameter as a key component of a functional trade-off between drought tolerance and drought avoidance (Bristiel et al. 2018). Under this trade-off, plants with large root diameters had higher water uptake and were better able to maintain growth under moderate drought but had lower survival under severe drought (i.e. lower drought tolerance). Our results suggest that accession “NM Dry” may be well suited to grow quickly while resources are abundant and avoid drought. This strategy may be particularly successful in the arid southwestern United States which is characterized by pulse precipitation regimes. Accurately characterizing droughts (Slette et al. 2019) and identifying which drought strategies will be most successful under particular precipitation conditions will be an important component of planning and implementing restoration projects in diverse drought-prone systems. Despite few differences in traits among accessions, drought had substantial effects on traits across all groups. Most of the shifts we observed (i.e. increased RTD; reduced SLA, SRL, number of root tips, total root length) provide evidence for E. trachycaulus plants adopting a more conservative resource use strategy under water stress. Few studies have assessed how traits related to root tissue construction (i.e. RTD, SRL) or architecture (i.e. number of root tips) change in response to water stress and results have largely been species specific (e.g. Hanslin et al. 2019; Larson et al. 2020; Lozano et al. 2020). Our results suggest that, in general, plants from different accessions of E. trachycaulus adjust root traits in similar directions in response to water stress, but the magnitude of responses may differ between accessions. As patterns in trait variability within species may vary from those observed among species (Grady et al. 2013), additional research that clarifies how intraspecific trait responses to drought affect plant performance is needed to more effectively apply traitbased approaches to restoration materials selection and development. July 2021 Restoration Ecology Relationships Among Traits, and Trait Plasticity Unlike in other studies (e.g. Larson & Funk 2016; Du et al. 2019), we did not find more or stronger trait correlations in our drought treatment compared to control. Rather, we found relatively consistent trait relationships across both developmental stages and treatments levels. In both control and drought conditions, traits associated with rapid above- and belowground resource acquisition (height, root length, root tips) were related. A recent study focused on woody species responses to drought stress found a similar positive relationship between height and root length (Li & Bao 2015). As number of root tips was also positively associated with these traits, it appears that extensive aboveground growth in E. trachycaulus is supported by similarly extensive and acquisitive root systems. The traits SRL and RTD were negatively related in all cases and related to RMR in drought conditions. Several studies (Holdaway et al. 2011; Kong et al. 2014) have found similar relationships between SRL and RTD, whereas others have found these traits to be decoupled (Craine et al. 2001; Kramer-Walter et al. 2016). Unlike leaves, which are tightly constrained along a primary functional axis of growth versus productivity (Wright et al. 2004), plants can construct roots with diverse trait combinations to function optimally in particular environments (Kramer-Walter et al. 2016; Bristiel et al. 2018). For example, Bristiel et al. (2018) found that perennial grasses with high RTD and high SRL roots were well suited to both maximize resource acquisition and tolerate drought. As water acquisition is anchored in root morphology and anatomy, understanding when and why these two traits are or are not coupled could advance our understanding of plant strategies for coping with drought. At both harvests and in both treatment conditions, the functional axis related to resource acquisition (RC1: height, root length, root tips) was related to drought resistance. This is unsurprising as plants require both above- (i.e. light) and belowground (i.e. nutrients, water) resources to accumulate biomass and traits related to rapid above- and belowground resource acquisition are often coupled (e.g. Comas et al. 2014; Freschet et al. 2015). Although this primary axis of variation was consistently related to aboveground biomass production, which secondary axes of functional variation were related to aboveground biomass production varied by harvest and watering treatment. At the 10-day developmental stage in both control and drought conditions, RC3 (i.e. SLA, root diameter) was related to aboveground biomass. This suggests that at this early developmental stage, plants with high SLA and low root diameter may be maximizing resource acquisition aboveground via thin and productive leaves and belowground with thin and absorptive roots. As in other species, this aboveground, belowground coordination may promote rapid growth and aboveground productivity (Comas et al. 2014). At the 24-day developmental stage in both control and drought conditions, all axes of trait variation were related to aboveground productivity, even though which traits were related to those axes varied. This suggests that, as they mature, plants may adjust the strategies they employ to maximize growth under resource-abundant versus resource-limited conditions. 9 of 12 �Drought cultivars underperform in drought settings In our study, root diameter was the least plastic trait between control and drought conditions. Working with four tree species, Kramer-Walter and Laughlin (2017) found root diameter to be among the least plastic traits in relation to nutrient limitation. Similarly, Hanslin et al. (2019) and Larson et al. (2020) found root diameter to vary little between control and drought conditions in grass species. As root diameter is highly phylogenetically constrained (Kong et al. 2014; Valverde-Barrantes et al. 2016) and an important component of plant hydraulic function (Wahl & Ryser 2000), plastic adjustments in this trait may be uncommon (but see Lozano et al. 2020). A deeper understanding of how root diameter influences the growth and survival of grass seedlings may provide valuable insight to its utility as a possible trait to screen for in the selection of native plant materials capable of thriving in resource limited conditions. The evidence we present here suggests that cultivars of E. trachycaulus bred for specific characteristics, in this case drought tolerance or resistance, do not necessarily possess traits that confer superior performance in water-limited conditions. Without obvious improvements in performance, reassessing whether widely available plant materials meet specific restoration goals is essential. Furthermore, potential negative impacts of using cultivars raise additional concerns about their ubiquitous use in restoration. Studies have found that the process by which native materials are selected and produced may result in unintentional selection for nonadaptive traits (Espeland et al. 2017) or lower variability in traits (Foxx & Kramer 2020). In addition, their introduction into sites with conspecifics may negatively affect the genetic integrity of local populations (Hufford & Mazer 2003). Dramatic shifts in the selection and production processes are needed to restructure current seed sourcing, collection, and production practices (Ladouceur et al. 2017). Albeit admittedly difficult, several recent advances indicate such shifts are already underway. For one, research about which traits improve restoration outcomes has grown rapidly in the last decade (e.g. Funk et al. 2008; Leger & Baughman 2015). In addition, rapid screening methods have been developed to aid in the identification of promising sources of restoration plant materials (Leger et al. 2020). Furthermore, sourcing and production practices aimed at retaining adaptive traits and genetic diversity have recently been refined (Espeland et al. 2017; Bucharova et al. 2019). All of these recent advancements will likely contribute to improved seed selection and production as the practice and science of restoration ecology continues to grow. 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Table S2: ANOVA results for effects of drought and accession on traits and biomass Figure S1: Trait means for each accession used in the study under control (circles) and drought (square) conditions at the 10-day harvest Figure S2: The first two varimax-rotated principal components (RC) plotted for traits measured at the 10-day harvests under (A) control and (B) drought conditions Figure S3: Boxplots show median, first and third quartile, and 95% confidence intervals of plasticity indices for each trait at the 10-day harvest Received: 16 February, 2021; First decision: 24 March, 2021; Revised: 5 April, 2021; Accepted: 9 April, 2021 Restoration Ecology July 2021 � https://cpw.cvlcollections.org/files/original/d6260e1ca12e696a24bcfea6387429ff.pdf 563d17270119372f620749ebb65a19b5 PDF Text Text Supporting Information Table S1: Information for the cultivar and wild accessions used in the study. Accession ID USDA Coordinates of original Annual GRIN ID collection Precipitation 'First Strike' 'Pryor' 'Revenue' 'San Luis' UT_Wet UT933-383 37.57111 -112.80916 750 mm CO_Mesic 39.578177 -107.25179 427 mm NMNW_Dry LLPMC-5 35.66973 -106.74295 260 mm 1 �Table S2: ANOVA results for effects of drought and accession on traits and biomass. Significant effects at the p <0.05 level are in bold and numerator and denominator degrees of freedom are provided for each model. Trait abbreviations are as follow: SLA: specific leaf area; SRL: specific root length; RTD: root tissue density; Tips: number of root tips. Trait responses 10-day Harvest 24-day Harvest Response Effects df F-value p-value df F-value p-value SLA Drought 13,67 1.462 0.231 13,78 10.282 0.002 Accession 1.895 0.095 1.700 0.132 Drought: Accession 0.297 0.936 0.603 0.727 Height Drought 13,97 9.685 0.002 13,90 69.072 <0.001 Accession 7.806 <0.001 6.305 <0.001 Drought: Accession 1.323 0.254 3.181 0.007 SRL Drought 4.468 0.037 13,85 13,88 40.380 <0.001 Accession 1.263 0.283 1.813 0.106 Drought: Accession 1.770 0.115 2.373 0.036 Length Drought 13.251 <0.001 13,96 13,90 22.561 <0.001 Accession 2.444 0.030 1.212 0.308 Drought: Accession 1.490 0.190 2.841 0.014 RTD Drought 0.520 0.473 13,88 13,88 14.891 0.000 Accession 0.659 0.683 1.311 0.261 Drought: Accession 1.507 0.185 0.879 0.514 Tips Drought 9.055 0.003 31.611 <0.001 13,96 13,88 Accession 1.521 0.179 1.144 0.344 Drought: Accession 0.887 0.508 1.920 0.087 Diameter Drought 0.585 0.446 13,96 13,90 12.618 0.001 Accession 5.367 <0.001 5.877 <0.001 Drought: Accession 1.899 0.089 0.534 0.781 Biomass Response Total Aboveground Belowground RMR Effects Drought Accession Drought: Accession Drought Accession Drought: Accession Drought Accession Drought: Accession Drought Accession Drought: Accession Biomass Responses 10-day Harvest df F-value p-value df 13,95 13,93 13,98 13,95 13,98 10.333 1.069 1.521 7.212 2.226 1.223 10.533 1.423 1.803 0.380 3.597 2.214 2 0.002 0.387 0.180 0.009 0.047 0.301 0.002 0.213 0.106 0.539 0.003 0.048 13,93 13,93 13,93 24-day Harvest F-value p-value 34.311 1.333 2.724 48.986 1.821 2.436 12.174 2.258 2.285 39.226 5.326 0.708 <0.001 0.250 0.018 <0.001 0.103 0.031 <0.001 0.044 0.042 <0.001 <0.001 0.644 �Height 12.5 a) Height [cm] 30 20 b) 10.0 B A Length [cm] 150 100 d) B A A A AB AB 40 20 0 Drought effect: p < 0.05, F = 4.47 Drought effect: p < 0.01, F = 13.25 Root Tips e) f) 0.20 75 Tips 0.15 0.10 50 25 0.05 0 Drought effect: p < 0.01, F = 9.05 Root Diameter g) B A A A Root Mass Ratio A A h) A RMR 0.3 0.75 0.2 ab ab a abb 0.50 ab ab abab bb ab ab ab Wild Accession UT Wet SRL [m g-1] RTD [mg mm-3] AB Drought effect: p < 0.01, F = 7.81 RTD Diameter [mm] A Root Length 60 c) 200 0.4 A 2.5 SRL 0.25 A 5.0 0.0 50 A 7.5 10 250 A Wild Accession CO Mesic SLA [mm2 mg-1] SLA 40 0.25 Accession Wild Accession NM Dry Cultivar San Luis Cultivar Revenue Drought Cultivar Pryor Drought Cultivar First Strike Wild Accession UT Wet Wild Accession CO Mesic Wild Accession NM Dry Cultivar San Luis Cultivar Revenue Drought Cultivar Pryor Drought Cultivar First Strike 0.00 Treatment First Strike Revenue NM Dry Pryor San Luis CO Mesic UT Wet Control Drought Figure S1: Trait means for each accession used in the study under control (circles) and drought (square) conditions at the 10-day harvest. Orange symbols show “Drought Cultivars”, blue symbols show “Other Cultivars” and gray symbols show “Wild Accessions”. Bars represent +/- standard error from the mean. Lowercase letters indicate significant differences at the p < 0.05 level among accessions and treatments. Uppercase letters indicate significant differences among accessions across both control and drought treatments. Model statistics for drought effects are provided. SLA: specific leaf area; SRL: specific root length; RTD: root tissue density. 3 �Control Drought 3 (A) 3 (B) RTD RMR 1 Diameter 0 Length Length Tips Tips HT SLA −1 Accession RTD 2 RC2 − 26% variance explained RC2 − 24% variance explained 2 Drought Cultivar First Strike RMR Drought Cultivar Pryor 1 Cultivar Revenue Length Tips 0 Cultivar San Luis HT SLA Wild Accession NM Dry Diameter −1 Wild Accession CO Mesic SRL −2 Wild Accession UT Wet −2 SRL −2 −1 0 1 2 RC1 − 35% variance explained 3 −2 −1 0 1 2 RC1 − 33% variance explained 3 Figure S2: The first two varimax-rotated principal components (RC) plotted for traits measured at the 10day harvests under (A) control and (B) drought conditions. Orange symbols show “Drought Cultivars”, blue symbols show “Other Cultivars” and gray symbols show “Wild Accessions”. Point show means and bars show standard error from the mean for each accession. HT: height; RMR: root mass ratio; RTD: root tissue density; SLA: specific leaf area; SRL: specific root length; Tips: root tips. Figure S3: Boxplots show median, first and third quartile, and 95% confidence intervals of plasticity indices for each trait at the 10-day harvest. Plasticity Index = | (maximum mean – minimum mean)/maximum mean |. Different letters denote significant differences in plasticity in traits at the p < 0.05 level. Trait abbreviations are as follow: SLA: specific leaf area; RMR: root mass ratio; SRL: specific root length; RTD: root tissue density; Length: total root length; Tips: number of root tips. 4 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Journal Articles Description An account of the resource CPW peer-reviewed journal publications Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Cultivars of popular restoration grass developed for drought do not have higher drought resistance and do not differ in drought-related traits from other accessions Description An account of the resource <span>Numerous functional traits have been identified as key contributors to plant performance under drought. However, many of these traits, specifically root traits, are rarely considered in the development of native plant cultivars. In this study, we assessed whether cultivars of the perennial grass </span><i>Elymus trachycaulus</i><span> (Slender wheatgrass) developed for drought differ in (a) drought resistance (i.e. a plant's ability to maintain aboveground biomass productivity under water deficit), (b) aboveground and belowground traits, and (c) trait responses to drought from other accessions (i.e. other cultivars, wild accessions). We also evaluated trait plasticity, assessed whether multivariate trait relationships varied between control and drought conditions, and determined which suites of traits are related to drought resistance. We worked with seedlings at two developmental stages to assess whether patterns vary ontogenetically. </span><i>E. trachycaulus</i><span> cultivars developed for drought did not differ from other accessions in drought resistance or traits related to drought-coping strategies. The effects of drought and accession on drought resistance, traits, and trait plasticity varied by developmental stage, but relationships among traits varied little between the two developmental stages. A primary axis of functional variation related to resource acquisition (plant height, root length, root tips) was consistently associated with drought resistance. However, which secondary axes were related to drought resistance varied by developmental stage and moisture condition. Our results suggest that traits and performance of commonly used cultivars ought to be reexamined to determine whether they are actually the best candidates for revegetation projects in specific contexts.</span> Bibliographic Citation A bibliographic reference for the resource. Recommended practice is to include sufficient bibliographic detail to identify the resource as unambiguously as possible. <p>Garbowski, M., D. B. Johnston, and C. S. Brown. 2021. Cultivars of popular restoration grass developed for drought do not have higher drought resistance and do not differ in drought‐related traits from other accessions. Restoration Ecology 29:e13415. <a href="https://doi.org/10.1111/rec.13415" target="_blank" rel="noreferrer noopener">https://doi.org/10.1111/rec.13415</a></p> Creator An entity primarily responsible for making the resource Garbowski, Magda Johnston, Danielle B. Brown, Cynthia S. Subject The topic of the resource Cultivars Drought strategies Functional traits Intraspecific trait variation Ontogeny Root traits Extent The size or duration of the resource. 12 pages Date Created Date of creation of the resource. 2021-04-15 Rights Information about rights held in and over the resource <div class="element"> <div class="element-text"><a href="http://rightsstatements.org/vocab/InC-NC/1.0/" target="_blank" rel="noreferrer noopener">In Copyright - Non-Commercial Use Permitted</a></div> </div> Format The file format, physical medium, or dimensions of the resource application/pdf Language A language of the resource English Is Part Of A related resource in which the described resource is physically or logically included. Restoration Ecology Type The nature or genre of the resource Article