Genotypic variation in traits linked to climate and … with home-climate temperature. Trait variation was largely explained by genotypic differences (H2 =0.33–0.85). Our results - [PDF Document] (2024)

Genotypic variation in traits linked to climate and abovegroundproductivity in a widespread C4 grass: evidence for a functionaltrait syndrome

Michael J. Aspinwall1,2,6, David B. Lowry1, Samuel H. Taylor1,3, Thomas E. Juenger1, Christine V. Hawkes1,

Mari-Vaughn V. Johnson4, James R. Kiniry5 and Philip A. Fay5

1Section of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA; 2Hawkesbury Institute for the Environment, University of Western Sydney, Richmond, NSW 2753,

Australia; 3Biology Department, Bowdoin College, Brunswick, ME, 04011, USA; 4USDA-NRCS Resources Assessment Division, Temple, TX 76502, USA; 5USDA-ARS Grassland Soil and

Water Research Laboratory, Temple, TX 76502, USA; 6Present address: Hawkesbury Institute for the Environment, University of Western Sydney, Hawkesbury Campus, Locked Bag 1797,

Penrith, NSW 2751, Australia

Author for correspondence:Michael J. AspinwallTel: +61 2 0498 599 747

Email: [emailprotected]

Received: 5 February 2013Accepted: 19 April 2013

New Phytologist (2013) 199: 966–980doi: 10.1111/nph.12341

Key words: C4, climate change, evolution,genecology, Panicum virgatum (switchgrass),physiology, polyploidy.

Summary

� Examining intraspecific variation in growth and function in relation to climate may provide

insight into physiological evolution and adaptation, and is important for predicting species

responses to climate change.� Under common garden conditions, we grew nine genotypes of the C4 species Panicum

virgatum originating from different temperature and precipitation environments. We hypoth-

esized that genotype productivity, morphology and physiological traits would be correlated

with climate of origin, and a suite of adaptive traits would show high broad-sense heritability

(H2).� Genotype productivity and flowering time increased and decreased, respectively, with

home-climate temperature, and home-climate temperature was correlated with genotypic

differences in a syndrome of morphological and physiological traits. Genotype leaf and tiller

size, leaf lamina thickness, leaf mass per area (LMA) and C : N ratios increased with home-

climate temperature, whereas leaf nitrogen per unit mass (Nm) and chlorophyll (Chl)

decreased with home-climate temperature. Trait variation was largely explained by genotypic

differences (H2 = 0.33–0.85).� Our results provide new insight into the role of climate in driving functional trait coordina-

tion, local adaptation and genetic divergence within species. These results emphasize the

importance of considering intraspecific variation in future climate change scenarios.

Introduction

A basic precept of evolutionary biology is that climate is a principaldriver of species distributions and local adaptation of populations(Turesson, 1922; Clausen et al., 1940; Rehfeldt et al., 1999). Evenso, we still lack a fundamental understanding of climate-driventrait coordination and climate-related physiological divergenceamong populations (Ackerly et al., 2000; Campitelli & Simonsen,2012; Wright & Sutton-Grier, 2012). It is also often unclearwhether intraspecific patterns are caused by plastic responses toenvironmental variation or genetically based differences (Sultan,2000; Etterson & Shaw, 2001; Chown et al., 2010). Therefore,examining intraspecific patterns of growth, morphology and func-tion in relation to climate will provide insight into physiologicalevolution and adaptation to climate, and is essential for predictingspecies responses to future climate (Arntz & Delph, 2001; Jump& Pe~nuelas, 2005; Albert et al., 2010).

Previous studies, across a range of species, have demonstratedintraspecific physiological variation associated with climate. Incommon garden studies population variation in net photosyn-thetic rates (ACO2), stomatal conductance (gs) and leaf nitrogen(N) have all been associated with climate of origin (e.g. tempera-ture, vapour pressure deficit, precipitation) (Oleksyn et al., 1998;Benowicz et al., 2000; Christman et al., 2008; Marchin et al.,2008). Intraspecific variation in intrinsic water-use efficiency(ACO2/gs or iWUE) has also been demonstrated and may reflectpopulation adaptation to arid conditions because high iWUEmay allow for carbon (C) fixation during water limitation(Comstock & Ehleringer, 1992; Voltas et al., 2008).

Climate may also drive functional trait divergence via indirectselection on correlated traits, resulting in genetic divergence ofentire trait syndromes (Falconer & Mackay, 1996; Lynch &Walsh, 1998; Ackerly et al., 2000; Geber & Griffen, 2003).Genetic variation in leaf mass per area (LMA), leaf carbon (C)

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and leaf N content, for instance, may co-vary with physiologicaltraits, reflecting physiological trade-offs and adaptation to localenvironments (Chapin et al., 1993; Reich et al., 2003). For exam-ple, low LMA (thin) leaves can exhibit high ACO2 and may bemore responsive to resource heterogeneity (Reich et al., 2003;Poorter et al., 2009), yet require greater N investment, havehigher respiration rates, and exhibit shorter life spans (Reichet al., 1998). Conversely, long-lived high LMA (thick) leavesoften exhibit lower N concentrations and ACO2, but demonstrategreater photosynthetic resource-use efficiency and stress resistance(Chapin et al., 1993; Kikuzawa, 1995). While general patterns oftrait coordination have been demonstrated across a wide range ofplant species (Reich et al., 2003; Wright et al., 2004), intraspe-cific patterns are typically less evident (Albert et al., 2011). Still,examining functional trait covariation among populations orgenotypes, in relation to their climate of origin, provides insightinto the factors that drive physiological divergence and localadaptation (Geber & Griffen, 2003; Albert et al., 2011).

Despite the adaptive importance of various functional traits,heritability estimates can vary widely, owing to environmentalsensitivity, developmental effects and trait co-variation (Donovan& Ehleringer, 1994; Geber & Dawson, 1997; Maherali et al.,2008). Yet, heritability estimates indicate how a trait mayrespond to selection pressures such as climate, and because traitsare often genetically correlated, a change in one strongly inheritedadaptive trait can result in broad changes to an entire trait syn-drome (Chapin et al., 1993; Ackerly et al., 2000). Therefore, her-itability estimates for an array of functional traits, measuredamong genotypes representing populations originating from dif-ferent climates may elucidate the environmental factors drivingtrait covariation and genetic divergence (Arntz & Delph, 2001).

Panicum virgatum (switchgrass) is an ideally suited plant spe-cies for examining climate-driven functional trait coordinationand genetic differentiation. This C4 NAD-malic enzyme(NAD-ME) type perennial bunchgrass is a key component of thetallgrass prairies of North America, and its geographic rangespans substantial variation in temperature, precipitation, photo-period and soil type. The species also has multiple uses includingforage, soil conservation, and bioenergy production (Parrish &Fike, 2005; Schmer et al., 2008). Panicum virgatum has shownstrong evidence of local adaptation; genetic differentiation inproductivity, flowering time and morphology have been primar-ily attributed to temperature (McMillan & Weiler, 1959;McMillan, 1965; Madakadze et al., 1998; Casler et al., 2004;Berdahl et al., 2005). Panicum virgatum populations have alsobeen generally classified in terms of habitat differentiation(Porter, 1966), with morphologically larger ‘lowland’ popula-tions originating primarily from the southern US, while ‘upland’populations largely occupy drier sites in the northern US. Ploidyalso varies among populations of P. virgatum across its geographicrange (Casler et al., 2011). Increased ploidy may confer an alteredphenotype capable of exploiting new ecological niches (Otto &Whitton, 2000; Maherali et al., 2009). Thus, P. virgatum is wellsuited for studying not only climate-mediated functional traitcoordination and genetic differentiation, but also physiologicaldifferentiation among ploidy types.

To understand how climate drives functional trait coordina-tion and genetic differentiation of populations, we grew nineP. virgatum genotypes in a common garden in central Texas andmeasured growth, morphological development, leaf traits andaboveground net primary productivity (ANPP). The nine geno-types originated from a broad range of temperature and precipita-tion environments in the Great Plains. We used broad-senseheritability (H2) estimates to determine the degree of genetic dif-ferentiation in all traits, compared morphology, leaf traits andANPP between ploidy types, and used multivariate analyses toexamine how genotype morphology, leaf traits and ANPP co-vary relative to the genotype’s home climate. We hypothesizedthat genetic differentiation in morphology, leaf traits and ANPPwould exhibit trends consistent with climate of origin; andwhole-plant growth and ANPP would show higher H2 than indi-vidual leaf traits, although a syndrome of key functional traitslikely to vary with climate (e.g. LMA, iWUE, leaf N) would alsoshow high H2.

Materials and Methods

Experimental facility

This study was conducted at a rainout shelter facility located nearTemple, Texas, USA (31°3′25.7″N, 97°20′50.9″W). Site eleva-tion is c. 199 m above sea level. The mean maximum temperaturein July–August is c. 35.0°C, and the mean minimum tempera-ture in December is c. 3.0°C. Soils are Austin silty clay (fine-silty,carbonatic, Udorthentic Haplustol), and are well-drained, withmedium to rapid runoff, and moderate to low permeability.

The facility consists of an 18.3 m9 73.0 m steel-framed rain-out shelter (Windjammer Cold Frame, International GreenhouseCompany, Danville, IL, USA) covered by a clear 240 lm poly-ethylene roof that transmits 90% of photosynthetically activeradiation (PAR). The 2.1 m high walls on the side of the shelterare open, and the eaves on the open ends of the shelter are 4.2 mhigh, maximizing air movement and heat dissipation.

The rainout shelter excludes natural rainfall from 245 m9 5 m plots arranged in four blocks. Within each block,plots are spaced 0.25 m apart, and blocks are spaced 2.76 mapart. Each plot is bounded by a vertical barrier of 1.84 mm thickpond liner (ethylene propylene diene monomer, Firestone Spe-cialty Products, Indianapolis, IN, USA) buried to a depth of1.2 m below the soil surface, which prevents horizontal sub-soilwater movement and root penetration from outside the plot. Thebarrier extends 10 cm above the soil surface and is supported by a5 cm9 10 cm wood frame that prevents surface water flow intothe plots from outside the shelter. Care was taken during con-struction to limit soil disturbance and compaction.

The plots were irrigated using 90° sprinklers (HunterHP2000, Hunter Industries Inc., San Marcos, CA, USA)attached to 1 m risers on the corner of each treatment plot. Thesprinklers were operated by a programmable controller (LEITXRC Series Ambient Powered Irrigation Controller, DIG Cor-poration, Vista, CA, USA).

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Genotypes

The study included nine different Panicum virgatum L. ‘geno-types’ representing different climatic origins and ploidy types(Table 1). We use the term ‘genotype’ to signify that these indi-viduals originated from vegetative propagation of a single plantand are representatives of the gene pool (population) present intheir locale. The use of these genotypes allows us to focus onbroader patterns of genetic variation in relation to climate, ratherthan within-population variation. Four of the genotypes (WBC,WIL, WWF and ENC) were propagated from wild collections.Two genotypes, KAN and VS16, were derived from cv Kanlowand cv Summer, respectively. NAS is an upland genotype fromnorthern Texas which has been used for land reclamation in thedry west. NOC is a USDA selection and while the lineage andexact collection location are unknown, it is an upland octoploidfrom the northern Great Plains. Genotype AP13 is an accessionof cv Alamo, a lowland ecotype, which is being sequenced by theJoint Genome Institute (JGI-DOE) as part of the switchgrassgenome project. VS16 is the primary upland ecotype beingsequenced by JGI. Genotypes KAN, VS16, NAS, NOC andAP13 all originated from prairie-remnant populations. Althoughsome genotypes are cvs, minimal domestication has left these cvsgenetically similar to the wild prairie remnant populations fromwhich they originated (Casler et al., 2007).

Climate

The genotypes originated from 27 to 41°N latitude, repre-senting a 11–22°C range in mean annual temperature (MAT;r = 0.99 with latitude) and a 650–1110 mm range in meanannual precipitation (MAP; not significantly correlated withlatitude, P = 0.25) (Table 1). We derived several more detailedmeasures describing the climatic origin of the genotypes(Table 1). These measures described temperature extremes andseasonality, and most were highly correlated with latitude

(r = 0.99) except for mean summer (May–September) precipi-tation (MSP; r = 0.63, P = 0.10). In addition, we calculatedthe annual heat–moisture ratio (AHM; ratio of mean annualtemperature to mean annual precipitation) and the summerheat-moisture index (SHM; ratio of the mean temperature inthe warmest month to summer precipitation). The AHM andSHM index the amount of precipitation available for plantgrowth (relative to atmospheric demand) (Rehfeldt et al.,1999); higher ratios indicate greater potential for water defi-cit. Both AHM and SHM decreased with latitude (r =�0.87and r =�0.71, respectively). These parameters indicate thatthe climate for genotypes from northern latitudes was coolerand less arid than for genotypes from southern latitudes(Table 1).

Propagation and establishment

Clonal replicates of each genotype were propagated via divisionand multiplication of rhizomes originating from a single plant.All replicates were grown outdoors in separate 3.79 l potsunder the same conditions and were transplanted into theplots on 3 March 2011 (day of year, DOY 62). All previousyear’s tillers were cut at 10 cm above the growth media surfaceand all transplants were dormant (i.e. no green tissue was visi-ble) at planting. The previous year’s tillers were counted andtested as a covariate in the growth analysis. Within each plot,two replicates of each genotype were planted at 1 m9 1 mspacing. Genotypes were assigned in a stratified random man-ner to positions within each plot, with one of each replicate inthe east and west halves of the plots, and with replicates neveradjacent to each other. All plots received identical irrigation sothat expression of genotypic differences could be explicitlystudied. Plots were initially well watered to facilitate establish-ment (March–May, 45–52 mmwk�1). Irrigation amounts thenapproximated the typical seasonal rainfall pattern for the studysite; June, July, August, and September irrigation amounts

Table 1 Ploidy, geographic origin and historical climate data for the nine Panicum virgatum (switchgrass) genotypes included in this study

Variable1 VS16 NOC KAN NAS WBC WIL AP13 WWF ENC

Ploidy 49 89 49 89 49 49 49 89 89Lat. (°N) 40.7 – 35.1 33.1 30.1 29.1 28.3 28.1 26.9Long. (°W) 95.9 – 95.4 96.1 98.0 98.2 98.1 97.4 98.1MAP (mm) 861 – 1045 1110 855 701 850 903 646MSP (mm) 518 – 503 468 397 345 408 410 315MAT (°C) 10.8 – 15.5 17.2 20.3 20.6 21.2 21.2 22.3MTCM (°C) �4.9 – 2.5 5.4 10.1 10.3 12.1 12.3 13.2MTWM (°C) 24.8 – 27.4 28.2 29.0 29.2 28.8 28.6 29.5TD (°C) 29.7 – 24.9 22.8 18.9 18.9 16.7 16.3 16.3DMT 138 – 70 58 17 30 13 9 10SHM (°C m�1) 48.0 – 54.4 61.0 71.3 84.1 64.1 56.9 78.5AHM (°C m�1) 12.5 – 14.8 15.5 23.7 29.4 24.9 23.5 34.5

Climate data (1971–2000) is from theNationalOceanic andAtmospheric Administration (NOAA)weather station closest to the genotype’s geographic origin.1MAP, mean annual precipitation; MSP, mean summer precipitation (May–September); MAT, mean annual temperature; MTCM, mean temperature ofthe coldest month; MTWM, mean temperature of the warmest month; TD, temperature differential (MTWM –MTCM); DMT, days with minimumtemperature < 0°C; SHM, summer heat: moisture index (MTWM : MSP); AHM, annual heat: moisture index (MAT : MAP).

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were 37.5, 30.0, 15.0 and 15.0 mm wk�1, respectively, for anannual total of 945 mm.

Ploidy determination

For genotypes whose ploidy was unknown (NOC, NAS,WBC, WIL, WWF, ENC), we determined ploidy by flowcytometry. Approximately 300–500 mg of young healthy leaftissue was combined in a small Petri dish with 1 ml PartecNuclei Extraction buffer mixed with 1 ll beta-mercaptoethanol(CyStain PI Absolute P reagent kit, Partec, M€unster, Ger-many). The tissue was chopped manually (5–7 min) with arazor blade while in the extraction buffer and allowed to sit forseveral minutes.

The extractant was passed through a Partec 30 µm Cell Tricsdisposable strainer placed over a 5 ml Falcon test tube. Stainingsolution (2 ml staining buffer, 12 ll propidium iodide (PI)stock, and 6 ll RNase, all from the CyStain PI Absolute Preagent kit) was then added to the extractant and the tubeplaced on ice in a dark cooler. The stained sample was analyzedwithin 30 min on a flow cytometer (FACSCalibur with Cell-Quest Pro software, BD Biosciences, Franklin Lakes, NJ, USA).By comparing the flow cytometry of each genotype with anAP13 tetraploid standard, we established the ploidy status ofeach genotype.

Growth and morphology

Genotype growth was assessed by recording the tiller number,canopy height (cm), and basal area (cm2) on all plants (n = 432)beginning at planting. Basal area was calculated assuming thearea of an ellipse based on the diameter of each transplant mea-sured in two perpendicular directions. Growth was measuredevery 8–9 d between DOY 70 and 144, and monthly thereafter.The presence of newly emerged panicles was recorded weeklyfollowing initial emergence. Between DOY 175 and 178, leafarea index (LAI, m2 m−2) for each plant was estimated fromceptometer (AccuPAR model LP-80, Decagon Devices, Inc.,Pullman, WA, USA) measurements at 10 cm height, taken intwo perpendicular directions through the center of the sward.

Tiller and leaf morphology was assessed at midseason (DOY209–216) by measuring leaf length (cm), leaf width (mm), tillerinternode length (cm) and internode diameter (mm). Measure-ments were made on three representative tillers per plant on allplants in one randomly chosen plot per block (n = 72), for a totalof 24 tillers per genotype. Leaf length and width (midway alongleaf) were measured on the second mature fully expanded (clearlydefined ligule) leaf from the tiller apex. Internode length anddiameter were measured between the first and second leaf at thebase of each tiller. Internode diameter was measured with a digi-tal caliper.

In mid-November (DOY c. 318), tillers were counted on allplants; each plant was harvested at 10 cm above the soil surface,dried at 65°C to a constant mass, and weighed to determineANPP (g m�2). Average tiller mass (g per tiller) for each plant

was calculated as ANPP divided by the number of tillers atharvest.

Leaf functional traits

Leaf gas-exchange and chlorophyll fluorescence were measuredmonthly (between DOY 136 and 293) on all plants within thesame four plots sampled for morphological characterization(n = 72). Leaf net CO2 assimilation (ACO2, lmol m�2 s�1), sto-matal conductance to water vapor (gs, mmol m�2 s�1), intrinsicwater-use efficiency (ACO2/gs or iWUE; lmol mmol�1), photo-chemical quenching of photosystem II (PSII) (qP, dimension-less) and efficiency of PSII (ΦPSII) were measured on one or twofully expanded, mature upper canopy leaves using a LI-6400portable photosynthesis system equipped with a modulatedchlorophyll fluorometer (6400-40) integrated into the cuvettelid (Li-Cor, Inc., Lincoln, NE, USA). The order in which plotsand genotypes were sampled was randomized. Measurementswere taken between 10:00 and 14:00 h approximating middayconditions: ambient photosynthetic photon flux density duringthis period was at least c. 1500 lmol m�2 s�1, which was theaverage midday PAR. Within the cuvette, PAR was maintainedat 1500 lmol m�2 s�1 using an actinic light source. The cham-ber CO2 supply was controlled at 405 lmol mol�1, and thesample [CO2] averaged 390� 6.7 lmol mol�1. At the begin-ning of each sampling period the cuvette block temperature wasset at ambient temperature. Leaf temperature was measureddirectly using the LI-6400 leaf thermocouple wire. Water vaporinside the chamber was not scrubbed so that relative humidityconditions inside the chamber tracked ambient conditions. Datawere recorded when ACO2, gs, intercellular [CO2] (Ci) and lightadapted fluorescence had stabilized. Fluorescence parameterswere calculated according to the built-in functions of the LI-6400 system.

The leaves sampled for gas exchange and chlorophyll fluores-cence were immediately removed with scissors and leaf laminathickness (lm) was measured on the base of each leaf using digi-tal calipers, carefully avoiding the leaf midrib. A small section(50 mg fresh mass) at the base of the cut leaf was removed fortotal chlorophyll (Chl) content (mg g�1 dry mass) determinationfollowing Wellburn (1994). Leaf area of the remaining leaf sam-ple was measured using a LI-3000A Portable Leaf Area Meter(Li-Cor). The sample was dried to a constant mass at 65°C, andleaf mass per unit leaf area (LMA; g m�2) was calculated as leafdry mass divided by leaf area. Dried leaf samples were groundinto a fine powder using a ball mill (SPEX SamplePrep 8000DMixer/Mill, SPEX SamplePrep LLC, Metuchen, NJ, USA), andstored under desiccation. Leaf C and N contents were measuredusing a combustion elemental analyzer (Flash 2000 Organic Ele-mental NC Analyzer, Waltham, MA, USA). Nitrogen per unitleaf area (Nl; mmol [N] m�2) was obtained as the product of Nper unit leaf mass (Nm; g kg�1) and LMA, multiplied by theatomic mass of N. Photosynthetic nitrogen-use efficiency(PNUE; lmol mol�1 s�1) was calculated as the ratio of ACO2

and Nl.

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Data analysis

All statistical analyses were carried out in SAS 9.2 (SAS Institute,Inc., 2002). Genetic variation in whole-plant growth over timewas examined using PROC MIXED with a repeated measuresmixed model in the form:

Yijkl ¼ lþ Ti þ Bj þ Pk þ Gl ðPkÞ þ TiBj þ TiPk þ TiGl ðPkÞþ BjPk þ BjGl ðPkÞ þ Rijkl þ eijkl

Eqn 1

where Yijkl represents the response variable (tiller number, canopyheight, basal area), Ti represents the ith day of the year, Bj repre-sents the jth block, Pk represents the kth ploidy level, Gl repre-sents the lth genotype from within the kth ploidy level. All otherterms represent the respective interactions and Rijkl and eijklrepresent the repeated measures term and residual, respectively.The number of tillers in the previous year was tested as a covari-ate. Effects were tested using Type III sums of squares. Leaf traitdifferences among genotypes were tested using the model:

Yijkl ¼ lþ Ti þ Zj þ Pk þ Gl ðPkÞ þ TiZj þ TiPk þ TiGl ðPkÞþ ZjPk þ ZjGl ðPkÞ þ Rijkl þ eijkl

Eqn 2

where Yijkl represents the response variable (ACO2, gs, iWUE, etc.)and all other parameters are the same as previously noted withthe exception of Zj, which is the effect of the jth plot. Lastly,genotype differences in leaf and tiller morphology were testedusing the model:

Yjkl ¼ lþ Zj þ Pk þ Gl ðPkÞ þ ZjPk þ ZlGl ðPkÞ þ ejklEqn 3

where Yjkl represents the response variable (leaf length and width;internode diameter and length) and the remaining effects in themodel are the same as stated previously. When genotype ortime9 genotype effects were significant (P ≤ 0.05), Tukey’sadjustment was used for pairwise comparison of genotype means.In all models, measurement date, ploidy, genotype, and theirrespective interactions were considered fixed effects. The block orplot effect and interactions with block or plot were consideredrandom effects.

To quantify the portion of growth, morphology and leaf trait(mid-summer only) variation attributable to genetic variation,broad-sense heritabilities (H2) were calculated as:

H 2 ¼ r2G=ðr2G þ r2B þ r2GB þ r2e Þ Eqn 4

where r2G, r

2B, r

2GB, and r2

e are the genotype, block, interac-tion and residual variances. All mixed models and H2 estimateswere calculated using PROC MIXED.

Pearson correlation coefficients were calculated to determinethe association between genotype growth and leaf trait means andthe climatic conditions from which the genotypes originated.

Correlation analyses were conducted in PROC CORR. All analy-ses were conducted at P ≤ 0.05 significance level.

We used principal components analysis (PCA) (PROC FAC-TOR) with orthogonal (varimax) factor rotation to reduce thetraits tested to a subset of variables that explained the majority ofthe observed variation, thereby reducing the influence of inter-trait correlations. We conducted the PCA using data collectedduring July (DOY 201) when the plants had reached peak mor-phological and physiological development. Derived variables orthose calculated as ratios, such as iWUE, PNUE and Nl, wereomitted to limit cross-confounding of factors. Parallel analysiswas used to determine the number of significant principal com-ponents (PC) to retain (Franklin et al., 1995; Peres-Neto et al.,2005). Multiple regression (PROC REG) was performed todetermine the climate variables that best explained the variationin genotype PC scores. Multicollinearity among the climate vari-ables was tested using variance inflation factors (VIF). Climatevariables with VIF > 10 were excluded from the model (Neteret al., 1996). Model selection was based on Akaike InformationCriterion (AIC) where lower AIC values indicate a more parsi-monious model.

Results

Genotypic patterns of growth, morphology, ANPP and leaftraits

Over the entire growing season, there was a significanttime9 genotype effect on tiller number, basal area and canopyheight, indicating that growth rates varied among genotypes(Table 2). The number of tillers in the previous year was a signifi-cant covariate for tiller growth (P < 0.0001) and basal area(P = 0.02), but not canopy height (P > 0.05). Genotypes originat-ing from more southern locations (e.g. ENC, WWF, WIL)showed the earliest tiller production (DOY 69) and continuedproducing tillers until the end of the growing season. Tiller pro-duction began later (DOY 88–94) and ended earlier for geno-types from more northern locations (KAN, NOC, VS16;Fig. 1a).

End of season canopy height was significantly differentamong the genotypes (Fig. 1b). These differences were positivelycorrelated with the genotypes’ home temperature conditions,and negatively correlated with TD and DMT, indicating thatgenotypes from warmer, less seasonal climates grew tallest(Table 3). Although end of season tiller production and basalarea were significantly different among genotypes (Table 2;Fig. 1a,c) these differences were generally uncorrelated withclimate (Table 3).

Panicle emergence of early flowering genotypes (AP13, VS16,NAS, NOC) was c. 2 months ahead of late flowering genotypes(ENC, WWF; Fig. 2). Genotype mid-summer (DOY 178) pani-cle emergence was not associated with climate (all P > 0.06).Given that AP13, a genotype representing a warm-climate popu-lation, showed earlier panicle emergence than expected, weexcluded AP13 from the analysis and found that, in this case,genotype mid-summer (DOY 178) panicle emergence showed

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negative correlations with MAT, MTCM and AHM (defined inTable 1), and positive correlations with TD and DMT (Table 3),indicating a tendency for cooler climate genotypes to begin flow-ering earlier.

Genotypes also showed significant morphological variation,with leaf and internode dimensions generally decreasing in morenortherly genotypes (Table 2; Fig. 3a,b). Genotype leaf and inter-node length decreased with MSP, and increased with MTWMand SHM (Table 3). Internode length increased with all threemean temperature variables and leaf width increased withMTWM (Table 3). These correlations suggested larger leaf andinternode dimensions for genotypes originating from environ-ments with warmer, drier summers.

Differences in ANPP among genotypes were highly significant(Table 2; Fig. 3c). Genotypes ENC, WWF, AP13, WIL andWBC were most productive (1195–1834 g m�2), followed byNAS and KAN (474 and 547 g m�2), and NOC and VS16 (130and 125 g m�2; Fig. 3c). Genotypic productivity differences wereclosely associated with climate of origin. Genotype ANPP and

tiller mass increased with MAT, MTCM, MTWM, SHM andAHM (r = 0.75–0.92; Table 3), and decreased with TD andDMT (r =�0.81 to �0.93) indicating that genotypes fromwarmer, less seasonal climates, were most productive (Table 3).The number of tillers in the previous year was a significant covar-iate for ANPP (P < 0.0001) but not tiller mass (P = 0.54).

Leaf trait differences among genotypes varied over time for alltraits except leaf thickness (Table 2; Supporting Information FigsS1, S2). With the exception of iWUE, gas-exchange and chloro-phyll fluorescence parameters peaked between DOY 166 and 201(Fig. S1). Physiological differences among genotypes were mostpronounced during this period. In comparison, genotype Nm, Nl

and Chl gradually decreased over time while LMA and C : Ngradually increased over time (Fig. S2).

Genotypes ENC and WWF showed the highest leaf thickness(301 and 265 lm, respectively), followed by WBC, AP13 andWIL (249–252 lm), NOC, KAN and NAS (177–233 lm) andVS16 (169 lm) (Table S1). When averaged over the entire sea-son, genotype iWUE, ΦPSII and C : N increased with genotype

Table 2 Degrees of freedom and F values for ANOVA of Panicum virgatum (switchgrass) whole-plant growth, morphology and leaf traits

Variable

Time Ploidy Genotype (ploidy) Time9 ploidyTime9 genotype(ploidy)

df F df F df F df F df F

ProductivityTillers 11,5045 1300.3 1,3 12.5 7,21 9.9 11,5045 48.1 77,5045 36.2Basal area 6,2934 828.3 1,3 202.6 7,21 18.0 6,2934 74.7 42,2934 10.6Canopyheight

6,2930 4495.6 1,3 2.4 7,21 204.3 6,2930 31.9 42,2930 61.8

ANPP1 1,3 2.8 7,21 47.9Tiller mass1 1,3 59.2 7,21 219.0LAI2 1,3 9.1 7,21 23.0

Morphology2

Leaf length 1,3 0.2 7,21 28.4Leaf width 1,3 2.1 7,21 32.5Internodelength

1,3 21.5 7,21 23.2

Internodediameter

1,3 9.1 7,21 10.7

Leaf traitsACO2 5,15 74.6 1,3 2.1 7,21 5.7 5,317 3.4 34,317 3.0gs 5,15 48.7 1,3 2.3 7,21 5.1 5,317 3.9 34,317 2.9iWUE 5,15 3.2 1,3 1.0 7,21 13.2 5,317 1.8 34,317 1.9PNUE 5,15 31.2 1,3 13.6 7,21 7.1 5,317 2.8 34,317 2.5ΦPSII 5,15 196.7 1,3 2.3 7,21 7.1 5,317 2.8 34,317 3.4qP 5,15 263.0 1,3 1.2 7,21 7.9 5,317 2.3 34,317 3.8LMA 5,15 78.9 1,3 83.3 7,21 20.5 5,317 0.8 34,317 1.6

Leaf thickness 5,15 12.7 1,3 6.1 7,21 23.1 5,317 1.8 34,317 1.1Nl 5,15 119.4 1,3 42.7 7,21 8.8 5,317 1.4 34,317 1.7Nm 5,15 214.2 1,3 0.7 7,21 33.7 5,317 1.9 34,317 2.5C : N 5,15 102.9 1,3 0.0 7,21 27.8 5,317 2.4 34,317 5.0Chl 5,15 15.8 1,3 1.1 7,21 21.8 5,317 1.7 34,317 2.2

F-values in bold are significant at P ≤ 0.05.1ANPP and tiller mass were measured at the end of the growing season.2LAI and morphological variables were measured at one time point (mid-summer).Leaf traits: ACO2, net photosynthetic rate; gs, stomatal conductance to water vapour; iWUE, intrinsic water-use efficiency; PNUE, photosynthetic nitrogen-use efficiency, ΦPSII, efficiency of PSII; qP, photochemical quenching of PSII; LMA, leaf mass area; Nl, nitrogen per unit leaf area; Nm, nitrogen per unit leafmass; C : N, leaf carbon : nitrogen ratio; and Chl, total leaf chlorophyll content.

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leaf thickness while genotype Nm and Chl decreased with geno-type leaf thickness (Fig. 4).

Genotype leaf trait means showed strong correlations withtemperature-related variables (Table 3), and none were corre-lated with MAP or MSP. Since temperature-related correla-tions were generally consistent over time, with the exceptionof May (data not shown), we only report correlations basedon genotype means pooled over time (Table 3). GenotypeiWUE, ΦPSII, LMA, leaf thickness and C:N increased withMAT, MTWM and MTCM, and decreased with TD andDMT (Table 3). Moreover, genotype iWUE and leaf thicknessincreased with both heat–moisture indices, SHM and AHM.Genotype Nm decreased with increased MAT and MTCM(Table 3). Therefore, a suite of leaf traits co-varied with thegenotype’s home-climate temperature conditions.

Ploidy

Octoploids produced more tillers and were larger in basalarea (174� 10 tillers and 888.3� 34 cm2, respectively) thantetraploids (115� 10 tillers and 503.7� 33 cm2, respec-tively) (Table 2). However, tetraploids produced larger tillers(6.9� 0.2 g per tiller) with longer internodes (17.8� 1.0 cm)than octoploids (5.7� 0.2 g per tiller and 15.4� 1.0 cm, respec-tively) (Table 2). Overall, ploidy had no effect on height growth orANPP (Table 2).

Most photosynthetic traits showed a significant time9 ploidyinteraction, indicating that ploidy effects on leaf physiology werenot consistent over time (Table 2). Even so, ploidy had a consistentsignificant effect on LMA and Nl (Table 2) with octop-loids showing higher LMA and Nl (84.2� 1.2 g m�2 and110.3� 1.8 mmol [N] m�2) than tetraploids (76.6� 0.9 g m�2

and 98.0� 1.4 mmol [N]m�2).

Broad-sense heritability

As expected, H2 estimates were higher for whole-plant growthand morphological traits (0.38–0.85) than for leaf traits(Table 4). Broad-sense heritabilities for photosynthetic traitswere moderate and ranged from 0.25 to 0.34 for gs and PNUE,respectively (Table 4). Other leaf traits tended to show higherH2 values and leaf thickness showed the highest H2 (0.60)(Table 4).

Principal components analysis and trait associations withclimate

Two PCs were identified which, cumulatively, explained 54.0%of the total variation in growth, morphology and leaf traits(Table 5). PC1, which represented a trait syndrome encompass-ing several growth, morphological and leaf functional traits,accounted for 33.6% of the total phenotypic variation. PC1 was

(a)

(b)

(c)

Fig. 1 Seasonal patterns of tiller production,height and basal area growth among Panicum

virgatum (switchgrass) genotypes from differentclimatic origins. Each symbol represents one ofthe genotypes identified in Table 1. Standarderror bars are not included in order to displaygenotype differences. DOY, day of year.

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positively correlated with canopy height, leaf and tiller size,LMA, leaf thickness and C : N, and negatively correlated withNm and Chl (Table 5; Fig. S3). PC2, which accounted for 19.9%

of the total variation, was positively correlated with ACO2, gs,ΦPSII and qP (Table 5; Fig S3).

Multiple regression analyses resulted in one climate variable,MTWM, that best explained the variation in genotype PC1scores (r2 = 0.93) (Fig. 5). Genotype PC1 scores increased ashome-climate MTWM increased. Moreover, there was a positiverelationship between genotype PC1 and genotype ANPP(r2 = 0.71, P < 0.01) indicating that genotypes originating fromclimates with the warmest summers were the most productiveand shared a common suite of functional and morphologicaltraits. By contrast, no combination of climate variables was effec-tive in explaining genotypic variation in PC2 scores (allP > 0.05), and genotype PC2 scores were not associated withgenotype ANPP (P = 0.19).

Discussion

Our results indicate that genetic divergence in growth, morphol-ogy and function among these P. virgatum genotypes is stronglyassociated with the temperature conditions in which the geno-types originated. Moreover, a syndrome of several functionallyadaptive traits with moderate to high heritabilities was correlated

Table 3 Correlations between Panicum virgatum (switchgrass) genotype productivity, morphology and functional trait means, pooled over time, andgenotype home-climate conditions (n = 8)

Variable MAP MSP MAT MTCM MTWM TD DMT SHM AHM

ProductivityTillers �0.23 �0.14 0.69 0.71 0.58 �0.73 �0.70 0.27 0.52Basal area 0.10 0.04 0.35 0.37 0.31 �0.38 �0.36 0.03 0.19Canopy height �0.57 �0.58 0.87 0.86 0.83 �0.85 �0.86 0.67 0.87ANPP �0.59 �0.54 0.92 0.92 0.82 �0.93 �0.90 0.63 0.86Tiller mass �0.57 �0.72 0.84 0.83 0.83 �0.81 �0.83 0.75 0.83LAI �0.62 �0.78 0.94 0.92 0.92 �0.91 �0.90 0.85 0.92Mid-summer panicle emergence1 0.60 0.38 �0.82 �0.84 �0.66 0.88 0.81 �0.46 �0.80

MorphologyLeaf length �0.41 �0.86 0.67 0.64 0.79 �0.58 �0.64 0.85 0.68Leaf width �0.20 �0.64 0.64 0.61 0.77 �0.55 �0.67 0.67 0.54Internode length �0.38 �0.77 0.77 0.75 0.86 �0.70 �0.78 0.81 0.69Internode width �0.19 �0.54 0.58 0.56 0.67 �0.51 �0.62 0.55 0.48

Leaf traitsACO2 �0.34 �0.42 0.53 0.52 0.51 �0.51 �0.56 0.41 0.48gs �0.06 0.04 �0.24 �0.25 �0.27 0.24 0.22 �0.14 �0.16iWUE �0.41 �0.60 0.91 0.90 0.91 �0.87 �0.89 0.71 0.81PNUE �0.05 �0.27 0.25 0.24 0.29 �0.21 �0.32 0.24 0.12qP �0.67 �0.35 0.24 0.22 0.15 �0.23 �0.23 0.33 0.46ΦPSII �0.37 �0.46 0.72 0.72 0.68 �0.72 �0.76 0.49 0.60LMA �0.48 �0.62 0.85 0.84 0.82 �0.83 �0.79 0.69 0.83

Leaf thickness �0.50 �0.64 0.95 0.94 0.92 �0.93 �0.92 0.72 0.89Nm 0.37 0.54 �0.74 �0.74 �0.69 0.75 0.70 �0.59 �0.62Nl �0.43 �0.45 0.65 0.65 0.63 �0.64 �0.59 0.51 0.73C : N �0.40 �0.59 0.93 0.93 0.88 �0.93 �0.91 0.67 0.77Chl 0.50 0.53 �0.66 �0.66 �0.59 0.67 0.60 �0.56 �0.68

Significant correlations are in bold.1Excluding genotype ‘AP13′. MAP, mean annual precipitation; MSP, mean summer precipitation (May–September); MAT, mean annual temperature;MTCM, mean temperature of the coldest month; MTWM, mean temperature of the warmest month; TD, temperature differential (MTWM –MTCM);DMT, days with minimum temperature < 0°C; SHM, summer heat: moisture index (MTWM : MSP); AHM, annual heat: moisture index (MAT : MAP). Leaftraits: ACO2, net photosynthetic rate; gs, stomatal conductance to water vapour; iWUE, intrinsic water-use efficiency; PNUE, photosynthetic nitrogen-useefficiency, ΦPSII, efficiency of PSII; qP, photochemical quenching of PSII; LMA, leaf mass area; Nl, nitrogen per unit leaf area; Nm, nitrogen per unit leafmass; C : N, leaf carbon: nitrogen ratio; and Chl, total leaf chlorophyll content.

Fig. 2 Variation in panicle emergence over time among Panicum virgatum

(switchgrass) genotypes from different climatic origins. Each symbolrepresents one of the genotypes identified in Table 1. DOY, day of year.

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with aboveground net primary productivity (ANPP). Overall,genotypic variation associated with climate, rather than ploidy,was the primary determinant of growth and functional strategies,and the trait syndrome we identified likely represents a continuumof traits reflecting adaptation to local climate, and would likely beimportant in determining the response of P. virgatum to climatechange.

Genotypic trait variation aligns with climate

The results supported our hypothesis that variation in genotypeproductivity, morphology, and functional traits would be corre-lated with climate of origin. Consistent with previous findings,P. virgatum genotypes from warmer climates began growth ear-lier, flowered later, and were more productive than genotypesfrom cooler locations. Thus, genotypes originating from warmerhabitats were more productive primarily due to a longer growingseason, brought about by warmer temperatures (Casler et al.,2007). Variation in vegetative and reproductive development inP. virgatum has long been associated with temperature (McMillan& Weiler, 1959; McMillan, 1965; Casler et al., 2004, 2007;Berdahl et al., 2005). However, warm climate genotypes origi-nated from a climate similar to our site which may have bolsteredtheir productivity. Nonetheless, we conclude that the observeddevelopmental differences among genotypes were primarilyrelated to the temperature conditions in which the genotypesoriginated. The observed pattern of genetic variation in growth,

development and productivity may indicate that genotypes maybe better described in terms of their position along a functionalcontinuum, rather than as distinct ecotypes (Porter, 1966; Casleret al., 2004).

It is possible that genotypic differences in flowering time andANPP may be associated with photoperiod sensitivity linked tothe genotype’s geographic origin (Sanderson et al., 1999; Casleret al., 2004). Flower initiation in some C4 grasses such asP. virgatum is dependent upon day length (Quinby, 1972), andmoving genotypes from northerly latitudes with long summerday lengths, to southerly latitudes with shorter summer daylengths, may induce flowering, thereby reducing the vegetativegrowth phase and ANPP (Van Esbroeck et al., 2003). We thinkthis mechanism is unlikely to be a confounding effect because ofthe strong correlations between genotype growth and develop-ment and climate of origin which reinforce the idea that climateof origin is the principal determinant of these traits inP. virgatum. Even so, genetic variation in photoperiod sensitivitywarrants further study.

As with phenology and productivity, the associations betweentemperature and genotype leaf and tiller morphology likely reflectadaptation to local climate. Often, genotypes adapted to aridconditions produce smaller leaves to reduce heat loads and con-serve water (Cunningham et al., 1999; Niinemets, 2001). How-ever, larger leaves have greater resistance to heat and mass transferthrough the leaf boundary layer, and differences in boundarylayer depth may influence iWUE (Parkhurst & Loucks, 1972;

(a)

(b)

(c)

Fig. 3 Mean (� SE) aboveground net primaryproductivity (ANPP) and morphological traitvalues for Panicum virgatum (switchgrass)genotypes originating from different climaticorigins where genotypes with the same letterare not significantly different at P ≤ 0.05. SeeTable 1 for details of the genotypes.

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Leigh et al., 2012). In particular, larger leaf size in warm-climategenotypes may be an adaptation that optimizes iWUE (Parkhurst& Loucks, 1972). Larger leaf size may also be associated withchemical and structural modifications required for growth andfunction under warmer temperatures or longer growing seasons(Niinemets et al., 2007). On the other hand, larger leaves requirevasculature of greater size and strength, which may explain whylarge-leaved genotypes produced larger tillers. Overall, theobserved morphological patterns are likely the outcome of trade-offs associated with temperature selection on structural and phys-iological traits (Parkhurst & Loucks, 1972).

Although we observed significant time9 genotype interactionsfor many leaf traits, most likely the result of phenological differ-ences, source-sink relationships, and environmental sensitivity(Long et al., 2006), we found evidence that genotypic patterns ofleaf trait variation are driven by climate, particularly temperature.Genotypes from the warmest environments showed a pattern of

leaf trait values known to confer higher tissue investment costs,yet enhanced resource-use efficiency and stress tolerance (Grime,1977; Chapin et al., 1993). By contrast, genotypes from coolerclimates showed a pattern of leaf trait values typical of plants withshorter growing seasons, higher physiological process rates andmore rapid tissue turnover (Chapin et al., 1993; Reich et al.,2003). These patterns align with broader patterns observedamong species (Reich et al., 2003; Wright et al., 2004), possiblyindicating that climate may influence interspecific and intraspe-cific functional trait divergence in a similar way (Albert et al.,2010).

One trait in particular, leaf thickness, showed consistent differ-entiation among genotypes (Fig. 4). Greater leaf thickness hasbeen interpreted as an adaptation to arid conditions (Wrightet al., 2004; Poorter et al., 2009). Thicker leaves may dampenpeak leaf temperatures, thereby reducing negative effects on pho-tosynthetic efficiency (Leigh et al., 2012). Indeed, genotype leaf

(a) (b)

(c)

(e)

(d)

Fig. 4 Relationship between Panicum

virgatum (switchgrass) genotype mean(� SE) leaf lamina thickness and genotypemean (� SE) (a) intrinsic water-use efficiency(iWUE), (b) nitrogen per unit leaf mass (Nm),(c) efficiency of PSII (ΦPSII), (d) leafchlorophyll (Chl), and (e) leaf carbon:nitrogen ratio (C : N).

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Tab

le4Gen

otypemea

ns(�

SE)an

dbroad

-sen

seheritab

ility

(H2)estimates

forproductivitymetrics,morphologicaltraitsan

dleaf

functionaltraitsam

ongninedifferentPanicum

virgatum

(switch-

grass)gen

otypes

Trait

NOC

VS1

6KAN

NAS

WBC

WIL

AP13

WWF

ENC

H2

Productivity

Tillers

128(7.3)

41(2.5)

101(6.2)

102(7.6)

127(5.3)

181(6.7)

129(5.2)

308(14.7)

158(6.1)

0.64

Basalarea

(cm

2)

475.5

(20.0)

512.7

(43.9)

399.6

(27.2)

1012.0

(66.0)

672.5

(31.1)

513.1

(24.5)

428.6

(21.3)

1153.7

(43.8)

903.8

(34.1)

0.52

Height(cm)

102.8

(2.0)

89.5

(1.7)

156.2

(3.4)

117.9

(3.5)

165.7

(1.9)

172.0

(1.9)

160.3

(1.8)

181.5

(2.2)

213.7

(2.7)

0.85

ANPP(g

m�2)

130.3

(20.8)

124.8

(8.3)

546.6

(49.9)

474.4

(47.2)

1408.0

(76.8)

1349.7

(55.4)

1194.9

(68.3)

1833.4

(91.7)

1719.4

(87.5)

0.66

Tiller

mass(g

per

tiller)

1.86(0.11)

2.10(0.12)

5.26(0.24)

4.54(0.36)

10.78(0.24)

7.48(0.21)

9.07(0.26)

5.93(0.15)

10.55(0.21)

0.81

Mid-sea

sonLA

I(m

2m

�2)

1.48(0.08)

0.65(0.05)

1.20(0.08)

1.38(0.12)

2.02(0.13)

2.32(0.11)

1.69(0.08)

2.07(0.11)

2.37(0.12)

0.38

Morphology1

Leaf

length

(cm)

43.3

(1.6)

33.1

(1.3)

43.9

(1.0)

50.1

(1.4)

61.2

(2.6)

54.6

(1.5)

49.2

(2.6)

38.1

(1.6)

59.9

(2.7)

0.74

Leaf

width

(mm)

10.1

(0.4)

8.8

(0.5)

13.8

(0.4)

12.9

(0.9)

16.8

(0.5)

14.5

(0.5)

13.3

(0.7)

12.0

(0.4)

15.5

(0.4)

0.72

Internodelength

(cm)

11.0

(0.4)

11.4

(0.9)

17.6

(1.0)

15.7

(1.3)

21.1

(0.9)

20.4

(0.8)

18.4

(1.3)

16.0

(0.9)

18.9

(0.9)

0.63

Internodediameter

(mm)

3.8

(0.1)

4.6

(0.2)

6.0

(0.2)

5.3

(0.3)

6.5

(0.3)

5.6

(0.2)

6.0

(0.3)

5.0

(0.2)

6.1

(0.3)

0.56

Leaf

traits1

ACO2(lmolm

�2s�

1)

28.3

(2.1)

25.9

(2.5)

30.4

(1.9)

26.3

(1.5)

37.5

(2.8)

26.2

(2.6)

36.3

(1.8)

30.5

(1.6)

31.5

(0.8)

0.30

g s(m

molm

�2s�

1)

203.7

(20.4)

203.4

(15.4)

167.2

(14.5)

173.0

(18.0)

255.1

(27.8)

138.3

(15.2)

205.7

(20.1)

162.4

(10.5)

205.1

(16.2)

0.25

iWUE(umolm

mol�

1)

0.143(0.01)

0.131(0.01)

0.186(0.01)

0.163(0.02)

0.156(0.01)

0.191(0.01)

0.181(0.01)

0.189(0.01)

0.161(0.01)

0.27

PNUE(lmolm

ol�

1s�

1)

206.7

(16.1)

247.9

(22.8)

284.6

(25.0)

214.1

(21.3)

357.7

(34.6)

228.7

(18.8)

326.1

(25.4)

266.0

(12.3)

259.2

(15.5)

0.34

ΦPSII

0.215(0.02)

0.211(0.02)

0.258(0.01)

0.218(0.01)

0.299(0.02)

0.232(0.02)

0.299(0.01)

0.261(0.01)

0.247(0.01)

0.40

qP

0.65(0.04)

0.62(0.05)

0.76(0.02)

0.62(0.02)

0.78(0.04)

0.68(0.03)

0.80(0.02)

0.74(0.02)

0.68(0.03)

0.32

LMA(g

m�2)

78.1

(1.3)

69.0

(3.0)

72.1

(3.0)

97.5

(10.3)

83.4

(3.5)

98.1

(3.5)

84.3

(1.3)

88.7

(1.7)

99.1

(4.8)

0.39

Leaf

thickn

ess(lm)

192.8

(10.8)

174.3

(7.9)

186.9

(12.0)

235.0

(13.7)

260.0

(10.2)

251.3

(15.2)

249.1

(8.9)

252.5

(12.6)

304.4

(6.8)

0.60

Nl(m

mol[N]m

�2)

137.1

(3.1)

104.2

(6.2)

109.0

(4.6)

129.4

(12.9)

107.0

(4.3)

113.8

(5.4)

113.4

(4.8)

114.9

(4.1)

124.2

(6.8)

0.19

Nm(g

kg�1)

24.7

(0.8)

21.2

(1.1)

21.3

(0.9)

18.8

(0.9)

18.2

(1.0)

16.5

(1.2)

18.9

(0.8)

18.2

(0.9)

17.7

(1.0)

0.43

C:N

17.8

(0.5)

20.3

(0.8)

20.9

(1.1)

23.0

(1.2)

24.1

(1.5)

27.0

(2.0)

23.3

(1.0)

23.9

(1.1)

25.3

(1.5)

0.33

Chl(mgg�1)

11.7

(0.4)

9.4

(0.6)

9.7

(0.8)

7.4

(0.6)

6.3

(0.5)

6.6

(0.5)

7.9

(0.4)

6.3

(1.1)

7.1

(0.4)

0.52

1Mea

nsan

dheritab

ility

estimates

areformea

suremen

tstake

nduringJulyonly.Le

aftraits:ACO2,net

photosyntheticrate;g s,stomatalconductan

ceto

water

vapour;iW

UE,

intrinsicwater-use

effi-

cien

cy;PNUE,

photosyntheticnitrogen

-use

efficien

cy,ΦPSII,efficien

cyofPSII;qP,photochem

icalquen

chingofPSII;LM

A,leaf

massarea

;Nl,nitrogen

per

unitleaf

area

;Nm,nitrogen

per

unitleaf

mass;C:N,leaf

carbon:n

itrogen

ratio;an

dChl,totallea

fchlorophyllcontent.

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thickness, iWUE and ΦPSII all increased with home climate tem-perature, suggesting that genotypes originating from habitatswith warmer and longer growing seasons possess multiple traitsassociated with photosynthetic resource-use efficiency (Geber &Dawson, 1990, 1997; Donovan et al., 2007). In comparison,Hartman et al. (2012) found no differences in ACO2 per unittranspiration among three latitudinally separated P. virgatumpopulations, yet significantly higher photochemical efficiency insouthern populations.

The negative association between Nm and temperature, a com-mon trend found within and among species, is thought to bedriven by temperature-related effects on plant stoichiometry,litter decomposition and N mineralization (Oleksyn et al., 1998;Reich & Oleksyn, 2004). Higher Nm in genotypes from coolerclimates is considered an adaptation that allows for continuedmetabolic activity and growth under low temperatures (Reich &Oleksyn, 2004). The negative relationship between leaf thicknessand Nm is consistent with the global leaf economics spectrum(Reich et al., 2003; Wright et al., 2004) which predicts thatthicker, high LMA leaves may require more upfront resourceinvestment, but are longer-lived and use resources more effi-ciently (Kikuzawa, 1995). This prediction matches the data forwarm-climate genotypes that showed early growth initiation, latepanicle emergence, thicker leaves, high LMA and high iWUE.

While climate of origin had a clear effect on genotype ANPP,morphology and leaf traits, ploidy effects were less consistent.Octoploids did show higher Nl which may reflect higher activitiesof N-rich photosynthetic enzymes (Warner et al., 1987). Yet,higher Nl in octoploids did not translate into higher photosyn-thetic rates, and tetraploids and octoploids showed significantvariation in leaf trait values over time. Although the reason forhigher Nl in octoploids is unclear, high Nl is sometimes found inplants from dry habitats and may be associated with higheriWUE (Wright et al., 2001), or an adaptation allowing for maxi-mum light utilization (Cunningham et al., 1999; Niinemets,2001). In agreement with previous results in P. virgatum, produc-tivity and physiological differences among these genotypes aremost strongly linked to their climate of origin, rather than theirploidy (Nielsen, 1947; Wullschleger et al., 1996; Casler et al.,2004, 2007; O’Keefe et al., 2013).

Important functional traits co-varied and were highlyheritable

The PCA indicated that LMA, C : N, Nm and Chl co-varied(PC1) (Table 5; Fig. S3), and although iWUE was not includedin the PCA, iWUE was correlated with the suite of leaf traits inPC1 (Fig. 4). Similar relationships between leaf thickness, LMA,iWUE, and leaf C and N to those we observed have been foundfor other C4 plants (Brown & Byrd, 1997; Arntz et al., 2000) aswell as for more restricted studies of P. virgatum (Byrd & May,2000). High LMA leaves tend to have lower concentrations ofN-based proteins but higher concentrations of C-rich compo-nents such as lignin and phenolics (Poorter et al., 2009), whichmay explain why genotype leaf thickness was positively correlatedwith leaf C : N ratio. Interestingly, the PCA revealed that leaf and

Table 5 Principal component factor loadings for productivity metrics,morphological traits and leaf traits in Panicum virgatum (switchgrass)

PC 1 PC 2

Eigenvalue 5.71 3.38% variance explained 33.6 19.9Productivity and morphologyHeight 0.78 0.22Tillers 0.13 0.15Basal area 0.17 �0.10LAI 0.48 0.20Leaf length 0.68 0.12Leaf width 0.68 0.38Internode length 0.69 0.21Internode diameter 0.60 0.33

Leaf traitsACO2 0.06 0.89gs �0.17 0.73ΦPSII 0.18 0.90qP 0.07 0.81LMA 0.62 �0.40

Leaf thickness 0.77 0.04Nm �0.83 0.22C : N 0.80 �0.25Chl �0.75 �0.05

Only data collected during July were used in PCA. Values in bold are thosewith eigen scores > 50. Leaf traits: ACO2, net photosynthetic rate; gs, sto-matal conductance to water vapour; iWUE, intrinsic water-use efficiency;PNUE, photosynthetic nitrogen-use efficiency, ΦPSII, efficiency of PSII; qP,photochemical quenching of PSII; LMA, leaf mass area; Nl, nitrogen perunit leaf area; Nm, nitrogen per unit leaf mass; C : N, leaf carbon: nitrogenratio; and Chl, total leaf chlorophyll content.

Fig. 5 Relationships between Panicum virgatum (switchgrass) genotypemean PC1 scores (� SE) and genotype’s home-climate mean temperatureof the warmest month (MTWM). Each symbol represents one of thegenotypes identified in Table 1.

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tiller morphology fall along the same axis as these key leaf traits,indicating a potential convergence of traits across levels of plantanatomical organization. These trait correlations could be theresult of pleiotropic effects of individual genetic loci on multipletraits (McKay et al., 2003; Remington & Purugganan, 2003).Alternatively, population structuring of alleles at different locicould each individually contribute to a component of the traitsyndrome.

This entire suite of traits was individually and collectively (inPC1) strongly correlated with aspects of the genotype’s hometemperature conditions, most strongly the mean temperature ofthe warmest month (MTWM). The same trait syndrome was alsoassociated with ANPP. Furthermore, the moderate to high H2 oftraits in PC1 provided evidence that home-climate temperature isthe major selective factor driving genetic divergence of coordi-nated traits in P. virgatum. Importantly, these H2 estimates mayindicate that when combined, coordinated sets of physiologicaland morphological traits may show significant responses to futureclimate thereby influencing the nature of any adaptive responsesin P. virgatum. Although genotypic expression may vary acrossenvironments (Campbell & Sorensen, 1978; Weinig et al.,2003), our intensive study under common garden conditionsprovides a strong basis for understanding how climate may drivephysiological divergence and local adaptation within species.

In broader terms, our study builds on classic genecology work(Turesson, 1922; Clausen et al., 1940; Campbell, 1979; Rehfeldtet al., 1999) and provides a more in-depth physiologically basedunderstanding of genetic divergence within species. Recent gene-cology studies have also demonstrated broad associations betweencoordinated sets of traits and climate (Johnson et al., 2010; Gradyet al., 2013). With climate change altering species phenology anddistributions (Cleland et al., 2006; Parmesan, 2006) and threat-ening species persistence (Jump & Pe~nuelas, 2005; Pe~nuelaset al., 2013), studies such as ours may be important for predictingspecies range shifts and defining seed transfer zones based onphysiological associations with climate (O’Neill & Aitken, 2004;Potter & Hargrove, 2012).

Importantly, our results support the concept of an adaptivetrait syndrome reflecting genotypic functional strategies deter-mined by climatic origin (Chapin et al., 1993), where longergrowing seasons necessitate conservation of resources, whichreduces tissue turnover rates, reduces the demand for additionalresources and increases stress resistance (Grime, 1977; Chapinet al., 1993; Reich et al., 2003). With extended periods of hightemperature and net declines in soil moisture expected in manyregions (IPCC, 2007; Karl et al., 2009), functional traits associ-ated with resource-use efficiency and stress tolerance may be keydeterminants of species growth and survival under novel condi-tions, as well as the sustainability of managed ecosystems (Kinget al., 2013).

On the whole, our results provide a basic understanding of therole of climate in determining functional trait coordination andgenetic differentiation. We propose that variation in growing sea-son length (i.e. phenology) among genotypes, which is princi-pally determined by the genotypes’ home-climate temperatureconditions, is the primary determinant of P. virgatum ANPP.

Functional traits associated with ANPP are not drivers of produc-tivity per se, rather they are traits having developed to match therequirements of a longer or shorter growing season. The syn-drome we define is likely to be a key determinant of P. virgatumresponse to environmental change because of its high degree ofgenetic differentiation and the importance of its composite traitsin resource uptake, use and turnover. These results provide newinsight into the way in which climate drives functional trait coor-dination, physiological evolution and local adaptation. Anthro-pogenic climate change has the potential to alter linkagesbetween environmental cues such as day length, temperature andgrowing conditions (e.g. frequency or occurrence of drought).These changes represent a clear challenge to the evolution of suchfunctional trait syndromes.

Acknowledgements

We thank T. Quedensley, K. Tiner, A. Naranjo, A. Gibson,L. Crosby, C. Steele, K. Baker, N. Johnson, and Y. Sorokin fortechnical support, and several anonymous reviewers for theirhelpful comments. M.A. thanks M. Tjoelker and P. Reich forsupport during the writing of this manuscript. USDA is an equalopportunity provider and employer. This material is based uponwork supported by the National Science Foundation (NSF)(IOS-0922457). Additional funding for D.B.L. was provided byUSDA NIFA postdoctoral fellowship (2011-67012-30696).P.A.F. acknowledges support from USDA-NIFA (2010-65615-20632).

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Seasonal variation in physiological traits amongP. virgatum genotypes from different climatic origins.

Fig. S2 Seasonal variation in leaf functional traits among differ-ent P. virgatum genotypes from different climatic origins.

Fig. S3 Results from principal components analysis (PCA) ofphenotypic traits in P. virgatum.

Table S1 Mean leaf lamina thickness of P. virgatum genotypesoriginating from different climatic origins

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