AJB Advance Article published on December 30, 2014, as 10.3732/ajb.1400234. The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1400234 American Journal of Botany 102(1): 000–000, 2015.

PLASTIC RESPONSES OF NATIVE PLANT ROOT SYSTEMS TO THE PRESENCE OF AN INVASIVE ANNUAL GRASS1

ALLISON J. PHILLIPS AND ELIZABETH A. LEGER2 Department of Natural Resources and Environmental Science, University of Nevada, Reno, 1664 N. Virginia Street, Reno, Nevada 89557 USA • Premise of the study: The ability to respond to environmental change via phenotypic plasticity may be important for plants experiencing disturbances such as climate change and plant invasion. Responding to belowground competition through root plasticity may allow native plants to persist in highly invaded systems such as the cold deserts of the Intermountain West, USA. • Methods: We investigated whether Poa secunda, a native bunchgrass, could alter root morphology in response to nutrient availability and the presence of a competitive annual grass. Seeds from 20 families were grown with high and low nutrients and harvested after 50 d, and seeds from 48 families, grown with and without Bromus tectorum, were harvested after ~2 or 6 mo. We measured total biomass, root mass fraction, specific root length (SRL), root tips, allocation to roots of varying diameter, and plasticity in allocation. • Key results: Plants had many parallel responses to low nutrients and competition, including increased root tip production, a trait associated with tolerance to reduced resources, though families differed in almost every trait and correlations among trait changes varied among experiments, indicating flexibility in plant responses. Seedlings actively increased SRL and fine root allocation under competition, while older seedlings also increased coarse root allocation, a trait associated with increased tolerance, and increased root mass fraction. • Conclusions: The high degree of genetic variation for root plasticity within natural populations could aid in the long-term persistence of P. secunda because phenotypic plasticity may allow native species to persist in invaded and fluctuating resource environments. Key words: adaptation; Bromus tectorum; competition; phenotypic plasticity; Poa secunda; Poaceae.

Phenotypic plasticity, or the expression of different phenotypes in different environments, is a common response of organisms to environmental variation (Bradshaw, 1965). Adaptive plasticity, wherein individuals increase fitness by changing their phenotype (Sultan, 2004; Ghalambor et al., 2007), may be important for the persistence of populations experiencing changes in climate, disturbance regimes, or facing new biotic interactions (Valladares et al., 2007; Matesanz et al., 2010). Understanding the degree of genetic variation within natural populations for plastic responses to environmental change is of increasing focus (Merilä and Hendry, 2014, and references therein), as the capacity for adaptive plasticity may be important for determining population persistence and range shifts (e.g., Phillimore et al., 2010). Introduced species present an opportunity to understand the importance of phenotypic plasticity for coping with novel conditions, and high plasticity is frequently posited as a strategy that favors success of introduced species (Richards et al., 2006; Pyšek et al., 2007; Davidson et al., 2011). On the other hand, native species can also experience dramatic changes in their

environment when new species invade. An increasing number of studies have demonstrated that the introduction of invasive species can dramatically change the trajectory of natural selection in wild populations, sometimes resulting in greater tolerance to invaders after selection (Strauss et al., 2006; Carroll et al., 2007; Oduor, 2013). However, no studies have examined the degree to which native plants can mediate the effects of exotic plant invasion via phenotypic plasticity. Plants are highly plastic organisms, readily altering their size, form, and function in response to changes in their environment (Bradshaw, 1965; Schlichting, 1986; Sultan, 2000). Many of these modifications are assumed to be adaptive because plants commonly respond to resource limitation by increasing allocation to structures involved in capturing the limiting resource (Bloom et al., 1985; Poot and Lambers, 2003; Hodge, 2009). For example, plants often allocate more biomass to roots when soil resources are limiting (Reynolds and D’Antonio, 1996) and can modify root form in different environments, changing root diameter, density, and architecture in response to soil resource availability (Campbell et al., 1991; Eissenstat, 1991; Fitter, 1994; Ryser and Lambers, 1995; Hodge, 2004). These types of root modifications may be important for native plant responses to disturbances like invasion, which can dramatically change the nature of soil resource distributions (Holmes and Rice, 1996; Ehrenfeld, 2003). One area where native plants are experiencing a dramatic shift in growing conditions is the Great Basin of the United States, where the invasion of exotic annuals, primarily Bromus tectorum L. (cheatgrass), is having dramatic effects on soil environments (Melgoza, et al., 1990; Norton et al., 2004; Rau et al., 2011). In this system, precipitation falls primarily in the fall and winter, with most plant competition occurring

1 Manuscript received 24 May 2014; revision accepted 1 December 2014. The authors thank O. Baughman, B. Wehan, S. Li, K. Badik, J. Greer, T. Larrieu, and A. Younie for their invaluable assistance with root washing and scanning and E. Espeland and E. Goergen for their roles in plant and seed collection. T. Albright, J. Cahill, S. Ferguson, M. Forister, J. Jernstedt, M. Matocq, F. Padilla, and D. Schwilk provided helpful comments on the experiment and manuscript. This research was funded by the University of Nevada, Reno, and the USDA-Hatch program. 2 Author for correspondence (e-mail: [email protected])

doi:10.3732/ajb.1400234

American Journal of Botany 102(1): 1–12, 2015; http://www.amjbot.org/ © 2015 Botanical Society of America

1 Copyright 2014 by the Botanical Society of America

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belowground during spring freeze–thaw cycles and continuing through midsummer, with active growth ceasing for all but the most deeply rooted shrubs as the summer progresses (Dobrowolski et al., 1990; Comstock and Ehleringer, 1992). Highly effective at capturing belowground resources, the winter annual B. tectorum develops longer, finer, and more branched root systems than seedlings of perennial species (Aguirre and Johnson, 1991; Arredondo and Johnson, 1999; Peek et al., 2005) and is extremely competitive with perennial seedlings (Booth et al., 2003; Humphrey and Schupp, 2002). With most competition occurring belowground, the ability to respond to the presence of competitive invaders via changes in root traits may be important for maintaining native species in invaded arid systems. Studies have shown that certain traits may allow for increased tolerance to B. tectorum competition, including early phenology, small size, reduced root diameters, and greater allocation to fine roots (Leger, 2008; Rowe and Leger, 2011; Goergen et al., 2011; Kulpa and Leger, 2013). Much is known about the inheritance of root system traits in crop and forage species (O’Toole and Bland, 1987) and other model organisms (Pacheco-Villalobos and Hardtke, 2012), including the degree of adaptive phenotypic plasticity in different cultivars (e.g., Liao et al., 2004) because this information can improve yield in agricultural systems. Much less is known about the inheritance of root traits in wild plants, where most research has focused on between-species (e.g., Crick and Grime, 1987; Arredondo and Johnson, 1999; Wahl and Ryser, 2000; Sultan, 2001; Hill et al., 2006) or between-population (Wilken, 1977; Heschel et al., 2004; Cordeiro et al., 2014) comparisons. Very little is known about the degree of root plasticity within wild populations (but see Schlichting and Levin, 1990; Sexton et al., 2002), though there is potential for within-population variation in adaptive phenotypic plasticity to play a large role in responses to disturbances such as invasion and climate change. Here, we investigated the ability of a native perennial plant to respond via phenotypic plasticity to the presence of a highly invasive species, looking specifically at plastic responses to changes in resource availability, both directly manipulated and as a result of growing with a highly competitive plant. We focused our work on Poa secunda J. Presl (sandberg bluegrass), a common perennial bunchgrass in the sagebrush steppe. With a growing-season phenology that overlaps strongly with B. tectorum, P. secunda is capable of significantly reducing B. tectorum biomass as an adult, more so than other perennial grasses (Goergen et al., 2011), but seedling establishment in sites dominated by B. tectorum is extremely challenging. The primary mating strategy for P. secunda is apomixis, and because most seeds are formed without sexual reproduction, populations have the potential for extremely low genetic diversity (Kellogg, 1990). However, apomixis can also maintain diversity within populations, preventing homogenization of genotypes due to outcrossing (Richards, 2003), and some populations of P. secunda are highly variable (Larson et al., 2001). The goals of our study were to (1) determine whether seedling root systems of P. secunda have similar phenotypic responses to two types of resource limitation, direct nutrient manipulations and competition from B. tectorum, (2) ask whether traits covary in response to resource limitation, and (3) identify which plastic responses were correlated with increased tolerance to reduced resources in a greenhouse environment. We expected to see correlated phenotypic responses to reduced resources, specifically increased root allocation under both low nutrients and competition (Reynolds and D’Antonio, 1996), an increase in

the production of fine roots, which are typically highly plastic and important for resource capture (Eissenstat, 1992), increases in specific root length (SRL, the ratio of root length to root mass), and root tip production, which is especially important for resource capture in resource-limited conditions (Poot and Lambers, 2003; Ryser, 2006), and expected these changes to be associated with increased tolerance to reduced resources. Finally, we were interested in quantifying the amount of genetic variation in phenotypic plasticity within and among populations, which is important for understanding the potential for adaptive evolutionary responses to environmental changes that affect resource availability. MATERIALS AND METHODS Forty individual adult plants of Poa secunda J. Presl (Poaceae) were transplanted from two sagebrush steppe locations in Nevada, McClellan Peak (39°14′21.30″N 119°44′34.70″W, 1750 m a.s.l.) and Bedell Flat (39°49′58.10″N 119°45′56.10″W, 1513 m a.s.l., sites described in detail in Goergen et al., 2011), to pots in the University of Nevada, Reno greenhouses in December 2008. Plants were grown in a common environment for one season to reduce the impact of maternal environment effects, and seeds were collected as they matured, from May through June 2009. Seeds were used from 48 families: 25 families from McClellan Peak and 23 from Bedell Flat. Nutrient manipulation experiment—In January 2010, 10 seeds from 20 families from McClellan Peak were sown into coarse sand in small pots (RLC4, 66 mL; Stuewe and Sons, Tangent, Oregon, USA) and held under controlled greenhouse conditions (4.4–26°C, 5–25% relative humidity). Pots were randomly assigned to either a low or high nutrient treatment. One 15 mL scoop of dry fertilizer (All Purpose Plant Food, 15-30-15 N-P-K, Miracle-Gro, Marysville, Ohio, USA) dissolved in 3.8 L of water was used for watering, 2–3 times per week for the high nutrient treatment; the low treatment was watered with a 1/4 strength solution for 30 d. Seedlings were harvested 50 d after emergence. Root systems were washed from sand, separated from leaves, and digitally analyzed using WinRhizo software (Regents Instruments, Siante-Foy, Quebec, Canada). Total root length, number of root tips, and the length of roots of different diameters were quantified for each plant. Initially, roots were analyzed in 10 different size classes, ranging from fine to coarse (Appendix S1, see Supplemental Data with the online version of this article), but for simplicity, analyses were also conducted on three root diameter size categories that had similar responses to treatments: 0–0.3 mm diameter, >0.3–0.5 mm diameter, and >0.5 mm diameter, corresponding to fine, medium, and coarse roots. Results from the simplified root categories are presented here, except when finer root divisions are needed to illustrate trade-offs among allocation to different root diameter classes. Roots and shoots were weighed separately after

TABLE 1.

F values, degrees of freedom (numerator df, denominator df), and significance of treatment, family, and interactive effects, from ANOVA to compare biomass and root responses, including root mass fraction (RMF), specific root length (SRL), and percentage allocation to three different root diameter categories, of Poa secunda to nutrient manipulations.

Response Biomass RMF SRL Root tips % Fine roots % Medium roots % Coarse rootsb

Treatment

Family

Family × Treatment

27.6(1, 20.7)*** 177(1, 20.3)*** 170.7(1, 20.7)*** 17.1(1, 20.2)** 136.3(1, 21.8)*** 168.6(1, 21.6)*** 52.5(1, 22.6)***

3.1(19, 19)* 2.1(19, 19)† 1.8(19, 19) 1.0(19, 19) 5.7(19, 19)** 5.3(19, 19)** 7.1(19, 19)***

1.6(19, 139)† 2.1(19, 139)* 2.2(19, 139)* 2.4(19, 139)* 1.0(19, 139) 1.1(19, 139) 0.8(19, 139)

Notes: ***P < 0.0001, **P < 0.001, *P < 0.05, †P < 0.10. bBox-Cox transformation before analysis.

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Fig. 1. Root mass fraction, specific root length (SRL), and number of root tips of Poa secunda plants produced under resource abundant (either high nutrients or grown alone) and resource limited (either low nutrients or grown with Bromus tectorum) conditions, with competition experiments harvested either early (after ~2 mo) or late (after ~6 mo). All differences between high and low resource responses are significant at P < 0.05 (Tables 1, 2). drying for 10 d at 60°C. Root mass fraction (root mass ÷ total plant mass) and specific root length (SRL, total root length in meters ÷ root mass in grams) were calculated for each plant. Higher SRL is typically, but not always, related to greater fine root production, as SRL is also affected by changes in root mass (e.g., Tjoelker et al., 2005). Allocation to each root diameter size class for each plant was calculated by dividing the length of a particular size class by total root length.

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Fig. 2. Differences in percentage allocation of Poa secunda plants to three root diameter size classes in response to (A) nutrient manipulation, (B) B. tectorum competition, harvested at ~2 mo old, and (C) competition, harvested at ~6 mo old. All differences between high and low resource responses are significant at P < 0.05 (Tables 1, 2).

Competition experiment—Forty seeds of 48 families of P. secunda were sown in December 2010 into both small (SC10, 164 mL; Stuewe and Sons) and large (TP49, 1.65 L) pots filled with a Nevada topsoil/perlite mix, with small pots used for plants that were harvested early. One seed of Bromus tectorum L. was sown in a randomly selected half of pots. Pots were misted once a day for the first 2 wk, then watered 1–2 times a week. An early harvest was conducted

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Fig. 3. Differences in mean family responses of Poa secunda to reduced resource environments, either direct nutrient manipulations or growing with B. tectorum competition for ~2 mo (early harvest) or ~6 mo (late harvest), with significance indicated as ***P < 0.0001, *P < 0.05, †P < 0.10 (family by treatment interactions, Tables 1, 2). from 15 February 2011 through 24 March 2011, with harvest timed exactly 67 d after emergence of each seedling, representing an early stage of plant establishment, when plants had, on average, about four leaves. A late harvest began approximately 6 mo after date of emergence on 31 May 2011, when B. tectorum was producing seeds and P. secunda was just starting to show signs of senescence. Plants were processed as in the nutrient experiment described, except that in competition pots, root systems of P. secunda and B. tectorum were carefully separated. Because of the time-consuming nature of the late season harvest, we could not separate root systems for all pots, and we completed this harvest for 39 of 48 families. As in the nutrient experiment, roots were analyzed first in 10 size categories, and the analysis was then simplified into three diameter size classes that corresponded to natural breaks in treatment responses (Appendix S1). Simplified root classes were: 0–0.4 mm diameter, >0.4–0.6 mm diameter, and >0.6 mm diameter, corresponding to fine, medium, and coarse roots. Analysis—ANOVA in the program JMP 9.0.2 (SAS Institute, Cary, North Carolina, USA) was used to determine whether treatments (either nutrient

addition or competition) had an effect on traits, whether families differed in measured traits, and whether families or populations differed in their plastic responses to treatments. Response variables were total biomass, root mass fraction, SRL, number of root tips, and percentage allocation to root diameter size classes. ANOVA models included the following factors: collection location (fixed factor, included in competition treatment models), family nested within collection location (random factor, included in competition treatment models), treatment (fixed factor), the treatment by family interaction, and the treatment by location interaction (included in competition treatment models), with these interactions interpreted as an indication of variation in phenotypic plasticity either among families or between populations (Schlichting, 1986). Variables were transformed as needed to improve distribution of residuals and homogeneity of variance assumptions; transformations are indicated in tables. When plastic responses are assessed, it is important to consider that plant traits can change in predictable ways during growth and development, a process referred to as ontogenetic drift (Evans, 1972; Wright and McConnaughay, 2002; Weiner, 2004). For example, some plants exhibit an initial high allocation

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Fig. 4. Two examples of active plasticity of Poa secunda in response to nutrient manipulations, showing the relationship between (A) percentage allocation of two root diameter size classes and (B) total biomass and the number of root tips, in high or low nutrient treatments. Relationships between traits differ significantly (P < 0.05) between treatments (trait by treatment interactions, see Appendix S2), indicating active increases in allocation to fine roots and root tip production in response to low nutrients. to roots during early seedling establishment, which decreases as plants grow, not as an active plastic response to an environmental stimulus but simply as a passive consequence of fixed ontogenetic changes that take place during growth and development (Gedroc et al., 1996; Weiner, 2004). Ontogenetic drift is especially important in experiments like the ones presented here, where the addition of nutrients or the presence of the strong competitor B. tectorum ensures that plants in some treatments are smaller than plants in other treatments and are thus likely to be at different developmental stages. To determine whether plant traits changed between treatment groups as a function of changing strategies (“active plasticity”), rather than just as a consequence of differences in developmental stages (“passive plasticity”), we used ANCOVA to test whether there were active or passive plastic changes in SRL, root tip production, and root mass fraction in response to treatments. Each trait was analyzed in a separate model, with a given trait and treatment as model factors and total biomass as the response variable. The treatment by trait interaction was included in the model, and the significance of this factor was used to determine whether the relationship between plant size and a given trait was on similar or different trajectories in different treatments. When interactions were significant, regressions were used to understand whether there was an increase or decrease in, for example, root tip production for plants of a similar size under a limited resource treatment. Similarly, to determine whether there were active changes in percentagae allocation to different root diameter classes, we used ANCOVA with one root diameter class as the response and the other class as a model

factor, again with treatment and treatment × trait interaction terms included as model factors, to determine whether allocation trade-offs between different types of roots were on different trajectories in different treatments. This analysis was conducted using the 10 root diameter size classes that showed the strongest inverse correlations, indicating potential trade-offs in allocation strategies. Finally, principal component analysis was used to determine whether suites of root traits changed together in response to reduced resource conditions and whether these correlations were similar between plants experiencing direct nutrient manipulation and competition with B. tectorum. We calculated mean trait values for each family when grown in each treatment and quantified familylevel plasticity for each trait by calculating the percentage change in the mean family value between the low and high resource environment (Valladares et al., 2006). Pearson’s correlations were conducted on these values for each experiment, and values were incorporated into a principal component analysis to visualize relationships among traits for the nutrient experiment, the early harvest, and the late harvest competition treatments. Positive correlations between tolerance to reduced resources, indicated by smaller reductions in biomass in low-resource environments, and plastic changes in other traits were taken as evidence that observed plastic changes may be adaptive. All data are presented as means ± SE, and significance is indicated by P < 0.05, though P < 0.10 are also indicated, so as to not overstate differences in plant responses to treatments.

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Nutrient experiment— Time to emergence ranged from 9 to 31 d after planting, with the majority of seedling emergence (approximately 91%) occurring between 11 and 17 d after planting. Seventeen seeds did not emerge. Nutrient treatments significantly affected all response variables (Table 1). In the low nutrient treatment, seedlings had less total biomass (low, 13.7 ± 0.6 mg, high, 18.7 ± 0.8 mg), a higher root mass fraction, higher SRL, and produced more root tips (Fig. 1). Percentage allocation to all three root diameter size classes changed in response to the nutrient treatment, with seedlings in the low nutrient treatment producing significantly more fine roots and significantly fewer medium and coarse roots (Fig. 2A). Variation among families—Families differed in biomass, percentage allocation to all three root diameter size classes, nearly differed in root mass fraction, but did not differ in SRL or the number of root tips (Table 1). The family by nutrient treatment interaction was significant for root mass fraction, with some families showing almost no change in allocation, but others increasing root mass fraction by up to 40% (Table 1). This interaction was also significant for SRL and root tip production (Table 1). All families increasing SRL under low nutrients, but differed in degree, and most families increasing tip production under low nutrients, but some showed reductions (Fig. 3). There were no significant family by treatment interactions for allocations to any of the three root diameter classes. The family by treatment interaction was nearly significant (P = 0.0559) for total biomass, with some families increasing performance by almost 60% under high nutrients, and others showing slight decreases. Plasticity and ontogenetic drift—There was a significant interaction between percentage root allocation to 10 different size diameter classes and treatment of five of 10 analyses (Appendix S2), indicating that plants actively shifted allocation patterns in response to nutrient differences in half of the analyzed relationships. In all five actively plastic changes, plants produced a greater amount of fine roots under low nutrients compared with

high nutrients relative to their size (Fig. 4A). Increases in root mass fraction and SRL were on the same trajectory in relation to total plant biomass (no significant treatment by trait interactions, all P > 0.10), indicating that these responses to low nutrients were passive changes and a function of differences in plant size between treatments. Changes in the number of root tips, on the other hand, reflected actively plasticity (F1, 176 = 33.3, P < 0.0001), with plants increasing the number of root tips produced at a given size under low nutrient conditions (Fig. 4B). Competition experiment— Seedlings emerged 8 to 43 d after planting, with approximately 94% of plants emerging after 10– 22 d. Thirty-eight plants of P. secunda did not emerge in the early harvest pots, and 56 did not emerge in the late harvest pots. Bromus tectorum emerged or was replanted in all competition treatment pots. Competition from B. tectorum strongly affected the plant size of P. secunda at both the early and late harvest (Table 2). Total biomass was reduced by 47% and 88% at the early and late harvest times, respectively (early: alone, 5.9 ± 0.2 mg, competition, 3.1 ± 0.1 mg; late: alone, 306.5 ± 6.0 mg, competition, 38.0 ± 1.2 mg). Similar to the response seen to low nutrients, root mass fraction, SRL, and percentage allocation to different root diameter size classes differed when plants were grown with B. tectorum (Table 2). As in the nutrient experiment, plants grown with competition had a greater allocation to roots, higher SRL, and a significantly greater percentage of fine roots and less medium and coarse roots than plants grown without competition in both the early and late harvests (Figs. 1, 2). In contrast to the nutrient experiment, there was a decrease in root tip production in the presence of B. tectorum, during both harvest times (Fig. 1). Plants of P. secunda from the two field locations differed in size and allocation patterns at the early harvest. Plants from McClellan Peak were larger, had more root tips, and had a lower SRL than plants from Bedell Flat, but differences in size decreased by the late harvest, and other differences were no longer evident (Table 2). Populations differed in the degree of plastic responses to the competition treatment of SRL and biomass at the early and late harvests, with greater changes in the

TABLE 2.

F values, degrees of freedom (numerator df, denominator df), and significance of factors from ANOVA of responses (RMF = root mass fraction, SRL = specific root length) of Poa secunda to competition from Bromus tectorum at (A) early harvest and (B) late harvest. Location

Location × Treatment

279.8(1, 48.6)*** 27.5(1, 49.1)*** 69.2(1, 48.5)*** 83.3(1, 47.8)*** 140.9(1, 47.9)*** 74.4(1, 47.5)*** 183.0(1, 47.7)***

56.7(1, 46.9)*** 2.0(1, 46.9) 6.7(1, 46.7)* 48.0(1, 46.9)*** 6.5(1, 46.6)* 3.6(1, 46.5)† 7.0(1, 46.8)*

5.71, 48.6* 1.5(1, 49.1) 8.3(1, 48.5)* 0.6(1, 47.8) 2.3(1, 47.9) 0.29(1, 47.5) 5.9(1, 47.5)*

3.0(46, 46)** 3.4(46, 46)*** 3.3(46, 46)*** 2.0(46, 46)* 3.1(46, 46)** 2.9(46, 46)** 2.1(46, 46)**

1.1(46, 707) 1.0(46, 707) 1.2(46, 707) 1.3(46, 715)† 1.2(46,715) 1.5(46, 715)* 1.3(46, 715)†

2415(1, 38,1)*** 161.7(1, 38.1)*** 495.4(1, 37.3)*** 813.1(1, 37.7)*** 604.4(1, 37.6)*** 18.5(1, 37.3)*** 749.8(1, 37.6)***

4.6(1, 37.4)* 1.5(1, 37.2) 1.1(1, 37.1) 2.4(1, 37.3) 0.01(1, 37.1)† 2.1(1, 37.1) 0.5(1, 37.1)

4.7(1, 37.8)* 1.0(1, 38.1) 7.1(1, 37.3)* 1.9(1, 37.7) 0.2(1, 37.6) 0.8(1, 37.3) 0.9(1, 37.6)

2.2(37, 37)* 6.1(37, 37)*** 2.5(37, 37)* 2.2(37, 37)* 8.2(37, 37)*** 3.9(37, 37)*** 6.7(37, 37)***

1.4(37, 592)† 1.0(37, 592) 3.4(37, 590)*** 1.5(37, 594)* 1.7(37, 594)* 3.2(37, 594)*** 1.7(37, 594)

Factor

Treatment

A) Early harvest BiomassL RMF SRL Root tipsL % Fine rootsb % Medium rootsb % Coarse rootsb B) Late harvest BiomassL RMF SRL Root tipsL % Fine roots % Medium rootsb % Coarse rootsb

Family

Notes: ***P < 0.0001, **P < 0.001, *P < 0.05, †P < 0.10; bBox-Cox transformation, Llog transformation before analysis.

Family × Treatment

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Bedell Flat population (Bedell Flat: 16% early and 60% late increase in SRL, 48% early and 88% late reduction in biomass; McClellan Peak: 9% early and 53% increase in SRL, 40% early and 86% late reduction in biomass). At the early harvest, the two locations differed in their degree of change in percentage allocation to coarse roots in response to the competition treatment (treatment by location interaction, Table 2), with plants from Bedell Flat again showing greater plasticity than plants from McClellan Peak, a difference that was not evident at the late harvest. Variation among families—In both the early and late harvests, families differed in all measured characteristics (Table 2). Differences in family responses to the competition treatment were especially apparent at the late harvest, when there were family by treatment interactions for SRL and root tip production (Fig. 3) as well as allocation to all three root diameter size classes (Table 2A); at the early harvest, families differed only in their production of medium diameter roots, though the family by treatment interaction for root tips was nearly significant (P = 0.09, Table 2B). Most families shifted traits in a similar way in response to competition, increasing SRL, decreasing root tip

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production, and increasing allocation to fine roots, though some families were more plastic than others (Fig. 3). Plasticity and ontogenetic drift—Five of 10 changes in correlations among root diameter classes were a result of active plasticity for the early harvest (Appendix S2). In all cases of active plasticity in the early harvest, plants produced a greater amount of fine roots under competition relative to control plants (Fig. 5). For the late-harvest, nine of 10 changes in correlations were a result of active plasticity (Appendix S2). Plants produced more fine roots for six correlations and more coarse roots for three correlations under competition relative to the control, reversing the direction of actively plastic changes from the early harvest in three cases where more coarse roots were produced in the competition treatment (Fig. 5). At the early and late harvests, changes in root tip production (early, F1, 803 = 56.5, P < 0.0001; late, F1, 662 = 4.3, P < 0.0001), and SRL (early, F1, 803 = 42.9, P < 0.0001; late, F1, 662 = 6.8, P = 0.0093) resulted from active plasticity, with smaller reductions in root tip production and higher SRL in the presence of B. tectorum than in similarsized plants growing alone. Changes in root mass fraction were the result of passive, rather than active, plasticity at the early

Fig. 5. Two examples of the relationship between percentage allocation of Poa secunda to root diameter size classes with and without competition with Bromus tectorum for the (A) early harvest and (B) late harvest. In the early harvest, plants grown in competition significantly (trait × treatment interaction P < 0.05, see Appendix S2) increased their allocation to fine roots, but by the late harvest, this pattern had reversed, with significantly lower allocation to fine roots in the competition treatment.

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harvest, but active at the late harvest, where plants that were grown alone increased their root mass fraction with size, while plants growing with competition maintained a constant allocation to root mass (F1, 662 = 89.3, P < 0.0001). Correlated shifts among traits—Correlations among trait changes showed both similarities and differences among all three experiments (Table 3, Fig. 6). The relationship between plastic changes in SRL and other traits differed most among experiments, as the change in SRL was highly correlated with increased tolerance (indicated by the degree of change in biomass), RMF, and root tip production in response to low nutrients, unrelated to tolerance at the early harvest but positively associated with RMF and root tip production and negatively correlated with tolerance at the late harvest, but unrelated to any other measures. Most consistent among all experiments, an increase in the amount of root tips (or, in the case of the competition experiments, a smaller decrease) was associated with increased tolerance to reduced resources (Table 3, Fig. 6). In all three experiments, plants were able to increase root tip production somewhat independently of shifts in root diameter, as these factors were typically orthogonal to changes in root diameter (Fig. 6). Increases in coarse root production were associated with increased tolerance in the early and late harvests, a pattern that was stronger in the late harvest (Table 3, Fig. 6). DISCUSSION Phenotypic plasticity is an important way for plants to mediate environmental variation, and adaptive plasticity may help organisms maintain viable populations in the face of extensive invasion. Focusing on a native perennial grass common in the

highly invaded sagebrush steppe, we asked whether native species were capable of responding to invasion via phenotypic plasticity. As predicted, P. secunda had similar responses to direct nutrient manipulation and competition with B. tectorum, responding to both conditions with changes in root morphology and allocation. Though responses to reduced nutrients and competition were similar overall, the correlations among these plastic shifts varied under different experimental conditions, with changes in SRL, in particular, differing in association across the three experiments, suggesting that plants of P. secunda have flexibility in their responses to different types of resource limitation. We observed considerable phenotypic variation among families for multiple traits, especially in the competition experiments, and families differed in their phenotypic responses to treatments, especially in the late harvest competition experiment. In addition, our two populations differed in their responses to the competition treatment, though these differences diminished by the late harvest. Every trait measured responded to our treatments, but some of these responses, such as root mass fraction in young plants, were due to ontogeny and differences in plant size (Evans, 1972; Wright and McConnaughay, 2002; Weiner, 2004), rather than active plasticity. We observed active increases in root tip production in response to resource limitation, a trait that was associated with increased tolerance in all experiments, as well as active increases in SRL and allocation to finer-roots for young seedlings grown under more stressful conditions, responses that have been observed in other species (e.g., Ryser and Lambers, 1995; Hill et al., 2006; Ryser, 2006). Older seedlings had slightly different root allocation patterns, with active increases in allocation to some coarse-root categories, another trait associated with increased tolerance to competition in older plants, and active increases in root mass fraction when grown

TABLE 3.

Pearson correlation coefficients and significance tests among values representing the percent change between family means in high and low resource environments for each trait (RMF = root mass fraction, SRL = specific root length), in (A) the nutrient experiment, and (B) the early and (C) late harvests in the competition experiment.

Response

ΔBiomass

ΔRMF

ΔSRL

ΔTips

ΔFine

ΔMedium

ΔCoarse

A) Nutrients ΔBiomass ΔRMF ΔSRL ΔTips ΔFine ΔMedium ΔCoarse

— 0.69** 0.63* 0.88*** −0.29 0.32 0.01

0.69** — 0.57* 0.77*** −0.45* 0.17 0.01

0.63** 0.57* — 0.69** −0.17 0.18 −0.29

0.88*** 0.77*** 0.69** — −0.44† 0.35 0.18

−0.29 −0.45* −0.17 −0.44† — −0.64** −0.53*

0.32 0.17 0.18 0.35 −0.64** — 0.28

0.01 0.01 −0.29 0.18 −0.53* 0.28 —

B) Early harvest ΔBiomass ΔRMF ΔSRL ΔTips ΔFine ΔMedium ΔCoarse

— 0.33* −0.25 0.68*** −0.05 −0.18 0.36*

0.33* — 0.45* 0.63*** 0.16 −0.15 −0.05

−0.25 0.45* — 0.32* 0.63*** −0.35* −0.65***

0.68*** 0.63*** 0.32* — 0.34* −0.38* −0.05

−0.05 0.16 0.63*** 0.34* — −0.80*** −0.73***

−0.18 −0.15 −0.35* −0.38* −0.80*** — 0.46*

0.36* −0.05 −0.65*** −0.05 −0.73*** 0.46* —

C) Late harvest ΔBiomass ΔRMF ΔSRL ΔTips ΔFine ΔMedium ΔCoarse

— −0.12 −0.33* 0.43* −0.14 0.05 0.45*

−0.12 — −0.07 0.04 0.16 0.19 −0.02

−0.33* −0.07 — 0.23 0.37* −0.03 −0.40*

0.43* 0.04 0.23 — 0.23 −0.09 0.17

−0.14 0.16 0.37* 0.23 — −0.15 −0.31†

0.05 0.19 −0.03 −0.09 −0.15 — 0.55**

0.45* −0.02 −0.40* 0.17 −0.31† 0.55** —

Notes: ***P < 0.0001, **P < 0.001, *P < 0.05, †P < 0.10.

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Fig. 6. Principal component biplots showing relationships among trait changes in plants of Poa secunda grown under resource abundant and resource limited conditions, manipulating either (A) nutrients or (B, C) competition with B. tectorum, with competition experiments harvested either early (after ~2 mo) or late (after ~6 mo). The amount of variation explained by each PC is in parentheses on the axis label, and dots represent each family’s phenotypic plasticity in response to reduced resources, calculated as the percentage change in mean family trait values. Points near the end of the “size” arrow are the most tolerant families, which experienced the smallest percentage change in biomass, and points near the end of all other arrows indicate families with the greatest increase (or smallest decrease, for root tips) in other depicted traits.

with B. tectorum, potentially indicating a shift from foraging to resource storage in older plants (Gregory, 2008). Though some actively plastic changes, such as an increased emphasis on root tip production, were consistently associated with increased tolerance to reduced resources under our experimental conditions, not all phenotypic plasticity is likely to be adaptive in a field setting. Maladaptive plasticity can persist in populations due to lack of reliable environmental cues, constraints that act directly on plasticity, and strong correlations among traits (Schlichting, 1986; DeWitt et al., 1998; Valladares et al., 2007; Auld et al., 2010). Species introductions can further complicate adaptive plasticity. Responses to, for example, novel predators, have been shown to elicit maladaptive responses in potential prey (Langerhans and DeWitt, 2002). Even very common plastic changes in plants, such as the ability to increase root mass fraction under resource-limited environments, are not always linked with increased competitive ability (Reynolds and D’Antonio, 1996). Field experiments with another perennial grass species common in the Great Basin, Elymus elymoides, indicated that seedling root traits such as SRL, total root length,

root mass fraction, and number of root tips strongly influenced seedling survival in sites invaded by B. tectorum (Atwater et al., 2015). A similar field test is ongoing with seedlings from the populations of P. secunda described here, transplanted back into invaded and uninvaded areas of their collection sites, which will allow us to determine whether observed plasticity is, in fact, adaptive under field conditions. In general, apomictic species have lower genetic diversity than outcrossing ones, and low genetic diversity has been observed in some apomicitic populations, apparently as a result of the dominance of a single clone (e.g., Paun et al., 2006). In contrast, in P. secunda, we observed considerable within and between-population variation, both in traits in general, and in plasticity. Fluctuating selection, frequency-dependent selection, and dispersal limitations are all factors that can maintain diversity in apomictic species (Roy, 1993; Van Dijk, 2003; Paun et al., 2006), and some combination of these processes may be operating to maintain diversity within P. secunda populations. In our experiment, we observed that plastic shifts in some traits, such as SRL, had different associates with tolerance in our three

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experimental conditions, and these types of divergent strategies under different types of resource limitation could contribute to the maintenance of diversity in plastic responses within populations. The existence of abundant variation in potentially adaptive traits may be positive for the long-term persistence of P. secunda, and there are different evolutionary trajectories these populations may take. If plasticity is associated with fitness costs, natural selection may favor fixed traits or genetic variation in nonplastic traits that induce no costs, leading to a reduction in family-level variation in the expression of plasticity (Pigliucci and Murren, 2003). Follow-up studies on these populations could provide information on how variation in plasticity may change over time in response to changes in growing conditions. Many of the traits investigated have been shown to be important for resource capture in other plant species. For example, fine root production and a high SRL influence foraging ability (Eissenstat, 1992; Hodge, 2004; Gregory, 2008), small changes in root diameter can have large effects on resource capture (Wissuwa, 2003), and plasticity in root allocation can affect competitive ability (Aerts et al., 1991). There are, however, many additional root traits that affect resource capture. We did not measure changes in the distribution of plant roots in the soil profile, root proliferation in response to neighbors, or root architecture, all of which can affect plant performance (Fitter, 1994; Pregitzer et al., 2002; Hodge, 2004; Gregory, 2008). None of these factors were directly examined here, but further studies could investigate how architecture, root proliferation in the soil profile, and other measures of allocation respond to the presence of competitive species, and how this may change beyond the seedling stage. Conclusions— The importance of plastic responses to novel interactions have been recognized in animal populations (e.g., Langerhans and DeWitt, 2002; Bourdeau et al., 2013), but despite their high capacity for plastic responses to changing conditions, the importance of phenotypic plasticity for maintaining native plant populations in the face of disturbances that reduce resource availability remain unknown. Ultimately, the longterm persistence of native perennial species like P. secunda in the Great Basin may depend on adaptive traits allowing plants to withstand changes in resource availability associated with the presence of competitive invasives (Leger, 2008; Leger and Espeland, 2010; Goergen et al., 2011; Rowe and Leger, 2011) and other environmental change. The ability to respond to belowground competition for limited resources through adaptive root plasticity may be one strategy that can promote survival of native plants in invaded arid systems. Poa secunda is an important species used in restoration in the Great Basin, yet some of the commercially available and widely planted varieties of P. secunda are particularly low in genetic diversity, with, for example, one common cultivar consisting entirely of one clone (Larson et al., 2001). Focusing instead on selecting diverse populations with genetic variation for traits such as plastic responses to resource variation for use in restoration may be key for the future of disturbed systems. LITERATURE CITED AERTS, R., R. BOOT, AND P. VAN DER AART. 1991. The relation between above- and belowground biomass allocation patterns and competitive ability. Oecologia 87: 551–559. AGUIRRE, L., AND D. A. JOHNSON. 1991. Root morphological development in relation to shoot growth in seedlings of four range grasses. Journal of Range Management 44: 341–346.

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