RESEARCH ARTICLE

AMERICAN JOURNAL OF BOTANY

Responses of two understory herbs, canadense and macrophylla , to experimental forest warming: Early emergence is the key to enhanced reproductive output 1

Marie-Hélène Jacques 2, Line Lapointe 2,5 , Karen Rice3 , Rebecca A. Montgomery 3, Artur Stefanski 3, and Peter B. Reich 3,4

PREMISE OF THE STUDY: Understory herbs might be the most sensitive form to global warming in forests, yet they have been little studied in the context of climate change.

METHODS: A fi eld experiment set up in , United States simulated global warming in a forest setting and provided the opportunity to study the responses of Maianthemum canadense and Eurybia macrophylla in their natural environment in interaction with other components of the ecosystem. Ef- fects of +1.7 ° and +3.4° C treatments on growth, reproduction, phenology, and gas exchange were evaluated along with treatment eff ects on light, water, and nutrient availability, potential drivers of herb responses.

KEY RESULTS: Overall, growth and gas exchanges of these two species were modestly aff ected by warming. They emerged up to 16 (E. macrophylla ) to 17 d (M. canadense ) earlier in the heated plots than in control plots, supporting early-season carbon gain under high light conditions before canopy closure. This additional carbon gain in spring likely supported reproduction. Eurybia macrophylla only fl owered in the heated plots, and both species had some aspect of reproduction that was highest in the +1.7 ° C treatment. The reduced reproductive eff ort in the +3.4° C plots was likely due to reduced soil water availability, counteracting positive eff ects of warming.

CONCLUSIONS: Global warming might improve fi tness of herbaceous species in deciduous forests, mainly by advancing their spring emergence. However, other impacts of global warming such as drier soils in the summer might partly reduce the carbon gain associated with early emergence.

KEY WORDS ; ; foliar nutrient concentrations; global warming; hardwood forest; phenology; photosynthetic rates; respiratory rates; understory light environment

Warming of the climate system is unequivocal ( IPCC, 2007 ). Th e carbon, energy, and nutrient cycling (Gilliam, 2007). Much less is herbaceous layer, with its high species richness, plays an important known about responses to warming of forest herbs compared with role in maintaining the functional integrity of temperate forest co-occuring larger trees. Climate change will require the migration, ecosystems through its interactions with the woody seedlings that acclimation, or adaptation of plant species because temperature is a determine, in part, the future canopy and through its role in crucial factor in determining the distribution of species ( Woodward and Williams, 1987 ). Since several forest understory species are very slow colonizers ( Verheyen et al., 2003 ), it is estimated that 1 Manuscript received 3 February 2015; revision accepted 11 September 2015. 2 Département de biologie and Centre d’étude de la forêt, Université Laval, Québec City, QC, their rate of migration might not match the rate imposed by , G1V 0A6; changes in climate. For these reasons, it is urgent to assess the eff ect 3 Department of Forest Resources, University of Minnesota, St. Paul, Minnesota 55108 of temperature on the reproductive output of herbaceous USA; and (Hovenden et al., 2008). So far, both positive and negative eff ects 4 Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, New South Wales, Australia have been reported on the reproductive output for the few herba- 5 Author for correspondence (e-mail: [email protected]) ceous species that have been studied ( Pearson and Shah, 1981 ; doi:10.3732/ajb.1500046 Hovenden et al., 2008 ; De Frenne et al., 2009 , 2010 , 2011 ); early

1610 • AMERICAN JOURNAL OF BOTANY 102 ( 10 ): 1610 – 1624 , 2015 ; http://www.amjbot.org/ © 2015 Botanical Society of America OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1611

spring fl owering species and more northern populations appear to Early senescence of herbaceous species subjected to a higher respond more positively to warming than summer fl owering and temperature regime was also observed ( Farnsworth et al., 1995 ; more southern populations ( De Frenne et al., 2009 ). Jochum et al., 2007), possibly indicating the development of a sink Global warming will also likely modify precipitation regimes al- limitation ( Lapointe, 2001 ). Another important component of plant though projection models do not agree, even at the regional scale. response to warming is physiological acclimation, which is the ad- For Minnesota, the PCM B1 model predicts an increased annual justment of photosynthetic and respiratory rates to improve plant rainfall with limited changes in summer precipitations, whereas performance at the new growth temperature ( Lambers et al., 1998 ). GFDL A1F1 scenario predicts a decrease in annual and in sum- Although photosynthesis and respiration are both known to be mer precipitations (Handler et al., 2014). Because both models temperature sensitive, there is growing evidence that both pro- predict increases in temperature during the summer and more cesses acclimate, sometimes even to the extent of homeostasis, in frequent extreme events, it seems reasonable to suggest that un- plants exposed to a moderate range of temperature (Campbell derstory species will experience more frequent and/or more se- et al., 2007; Sendall et al., 2015). We conducted a study on the eff ect vere drought in the future. Such changes can have direct adverse of experimental warming on two understory species widespread eff ects and might dampen any positive impact of warming on ni- in the southern boreal forests of : Maianthemum trogen supply through increased soil net nitrogen mineralization canadense Desfontaines and Eurybia macrophylla (L.) Cassini. Th e (Griffi n, 1981; Rustad et al., 2001; Schimel et al., 2007; Manzoni study system, the B4WarmED experiment, is a free-air warming et al., 2012). facility used to study the eff ect of a warming climate on the regen- Whether growth responses to warming of understory herbs dif- eration of woody species ( Reich et al., 2015 ; Rich et al., 2015 ). fers from that of sympatric trees remains unclear. So far, the general Because the warming system is installed in a forest, it allows us to response of plant productivity in climate warming experiments study not only the eff ect of warming on the two species of interest, has been positive, but because many of the studies used in meta- but also its interaction with the soil system and other herbaceous analyses were conducted in Arctic ecosystems, these results are not and woody species. We tested the following series of hypotheses necessarily indicative of temperate forest herbs ( Arft et al., 1999 ; (Fig. 1). Rustad et al., 2001 ; Dormann and Woodin, 2002 ; Walker et al., Direct and indirect eff ects of warming on the ecosystem compo- 2006 ). A recent meta-analysis used the plant functional group ap- nents will likely translate into changes in growth and reproduction proach and included 127 studies, many at mid-latitudes ( Lin et al., of the studied species. Some of these eff ects are expected to improve 2010 ). Th at study found that the increase in biomass of herbaceous growth and reproductive output of the herbaceous species, e.g., in- species (+5.2%) was smaller than the increase of woody species creased nutrient availability and earlier emergence, whereas factors (+26.7%) although much of this difference was explained by the such as lower water availability or lower light due to shading by fact that the eff ect on herbaceous species from studies of lower lati- taller plants are expected to negatively affect the two studied tudes was negative. Milder eff ects or negative eff ects of warming on species. Furthermore, some factors might have no net eff ect on car- herbaceous plants, where they cohabit with woody species, might bon gain, for instance, acclimation of the gas exchange rates. Spe- be explained through competitive interactions with plants of higher cifi cally, the objectives of the current study were to quantify the strata. Th e smaller size of herbaceous plants makes them more vul- impact of 3 years of warming on M. canadense and E. macrophylla nerable to shading from taller plants ( Castro and Freitas, 2009 ), and growth and reproduction. Furthermore, we tested if light availabil- their shallow roots make them more vulnerable to drier soil condi- ity in the understory both in the spring and in the summer, plant tions ( Jackson et al., 1996 ). phenology, leaf photosynthetic and respiratory rates and/or plant Climate change is expected to lengthen the growing season in nutrient concentration were modulated by the warming treatments. temperate climates. Earlier emergence of spring herbs following an Th is study quantifi es the growth response of two herbaceous species increase in temperature has been reported (Farnsworth et al., 1995; to warming and helps to unravel some of the factors and mecha- De Frenne et al., 2011 ) and can be a great advantage since many nisms that contribute to this response. understory herbs produce most of their photosynthates before the closure of the canopy (Bazzaz and Bliss, 1971; Brown et al., 1985; Lapointe, 2001). However, this advantage can be reduced if trees MATERIALS AND METHODS leaf out earlier, thus reducing the duration of the high light period for herbaceous growth (Routhier and Lapointe, 2002). Herbaceous Biology of the studied species — Maianthemum canadense (false species that emerge during or aft er canopy closure exhibit limited lily of the valley; Liliaceae) is a rhizomatous clonal plant 5 to 20 cm changes in phenology ( Ishioka et al., 2013 ). Th e response of the high. Th e species is found in moist woods and thickets where it phenology of the overstory to warming is more complex than that sometimes forms continuous mats ( Gleason and Cronquist, 1963 ). of the herbaceous layer since trees and shrubs are also infl uenced by It is an early bloomer and thus emerges and early in photoperiod or require a certain degree of winter chilling to be- the growth season. Th e mature slowly throughout the sum- come temperature sensitive in spring (Körner and Basler, 2010). mer to become, in late summer, bright red berries sometimes borne Temperature sensitivity of tree leaf emergence also decreases with by a plant with totally senesced leaves (M. H. Jacques, personal ob- an increase in latitude ( Menzel et al., 2006 ; Wang et al., 2015 ). Th e fact servations). Th e species is common in deciduous and boreal forests that tree phenology is modulated by many factors could partly ex- from northern British Columbia and Alberta to southeastern Mon- plain why spring emergence in a time series study yields stronger tana and Wyoming ( LaFrankie, 2003 ). Its range continues south- advancement trends in herbaceous than in tree species ( Ge et al., ward along the Blue Ridges to . 2015 ). Yet, variability among species response within a same lati- Eurybia macrophylla (large leaf ; Asteraceae) is a rhizomatous tude or community precludes predictions of changes in spring phe- clonal plant larger than M. canadense . It oft en forms dense mats ex- nology at the species level ( Parmesan, 2007 ). cluding other species ( Schulz and Adams, 1995 ). It is found in mixed 1612 • AMERICAN JOURNAL OF BOTANY

were historically higher at the south- ern site, the growing season tempera- tures during the experiment have been similar at the two sites ( Rich et al., 2015 ; Table 1). Nevertheless, growing season was longer (226 vs. 211 d) at the more southern site. Within each site, half of the plots are located in a mixed forest stand about 40–60 yr old, mainly composed of aspen, birch, and fi r trees, and the other half are located in a section of the forest that has recently been clear-cut. Clear-cut areas are invaded by herba- FIGURE 1 The network of hypotheses being tested in the current study. ceous species commonly associated with fi elds or disturbed areas. For this temperate forests and tolerates a wide range of light regimes, from reason, the current study was restricted to plots located in the intact shady undergrowth to wide sunny gaps ( Schulz and Adams, 1995 ). It forest stands, to focus on the impact of global warming on the natu- fl owers from late summer to late autumn ( Marie-Victorin, 1935 ) and ral understory layer. produces winged achenes. Th e species is common in northeastern deciduous or mixed forests. Its range extends from the southern bo- The warming technique — Th e B4WarmED experiment combines real forest of Eastern Canada to northern and along the Blue two techniques: IR lamps and buried heating cables (Rich et al., Ridges (Brouillet, 2006). It becomes rare at the western edge of its 2015 ). Th is combination overcomes some of the main disadvan- range (, , , and ). tages of these techniques taken individually. It prevents the soil from being not properly warmed by the lamps by compensating Study site — Th e study was conducted within the B4WarmED ex- with heating cables and avoids the decoupling of belowground and periment (Boreal Forest Warming at an Ecotone in Danger). Th is aboveground parts by heating them simultaneously. One can thus experiment was established to evaluate, through experimental achieve a more realistic simulation than if heating only the below- warming of forest plots, the extent to which, and the mechanisms ground or the aboveground parts ( Rich et al., 2015 ). whereby, projected global warming could alter the composition of IR lamps of 1000 W are located around the perimeter of each tree species of the southern boreal forest (Reich et al., 2015; Rich plot at an angle of 45° . Dummy lamps are used for the control plots. et al., 2015 ). Th e heating cables, buried to a depth of 10 cm, are spread 20 cm Th e B4WarmED experiment has two locations. Th e site located apart. Th e plots are connected to a monitoring system that main- at the Cloquet Forestry Center (CFC) is at a slightly lower latitude tains a constant temperature diff erential relative to control plots by (Cloquet, MN, 46 ° 31′ N, 92° 30′ W, 386 m a.s.l., 4.5° C mean annual turning on and off lamps and cables as needed (Table 1, see Rich temperature [MAT], 807 mm mean annual precipitation [MAP]) et al., 2015 for more information). Th e heating system is in operation than the site located at the Hubachek Wilderness Research Center about 8 mo a year; it is turned on when the temperature is ≥ 1 ° C for (HWRC) (Winton, MN, 47° 55 ′N, 92 °30 ′W, 453 m a.s.l., 3.0° C MAT, 5 consecutive days in spring and turned off when the temperature 722 mm MAP). Although mean annual and seasonal temperatures is ≤ 1° C for 5 consecutive days in autumn.

Experimental design — Th e experimen- TABLE 1. Average (± SE) aboveground temperature (° C), soil temperature (° C), and soil moisture (%) for the tal design is a generalized split-plot diff erent treatments at each site for the growing season 2011, i.e., from 9 April to 21 November at CFC and from with the site as the main plot and the 21 April to 18 November at HWRC and for the growing season 2012, i.e., from 21 March to 24 November at CFC treatment as the subplot. Th ere are two and from 27 March to 16 November at HWRC. Results of ANOVA and of a posteriori contrasts for these data are warming treatments, +1.7° C and +3.4 ° C, presented in Table 2 . the ambient temperature control plots, Average aboveground Average soil plus one treatment at ambient temper- Year Site Treatment temperature (° C) temperature (° C) Average soil moisture (%) ature with buried but inactive cables 2011 CFC Ambient 11.0 ± 0.08 11.5 ± 0.06 22.0 ± 0.94 to control for the eff ect of soil distur- CFC +1.7° C 12.6 ± 0.04 13.3 ± 0.05 19.2 ± 1.09 bance. Data collected in the plots of CFC +3.4° C 13.9 ± 0.04 14.9 ± 0.04 18.4 ± 0.71 both treatments at ambient tempera- HWRC Ambient 12.7 ± 0.10 12.6 ± 0.10 24.9 ± 3.28 ture show no significant differences ° ± ± ± HWRC +1.7 C 14.5 0.08 14.4 0.05 21.9 2.16 indicating that the disturbance of the HWRC +3.4° C 16.0 ± 0.11 16.1 ± 0.05 20.8 ± 1.91 2012 CFC Ambient 9.8 ± 0.09 10.8 ± 0.11 17.3 ± 0.23 soil had very little impact on the pro- CFC +1.7° C 11.5 ± 0.04 12.4 ± 0.11 15.8 ± 0.23 cesses under study (unpublished data, CFC +3.4° C 13.1 ± 0.05 13.7 ± 0.08 16.0 ± 0.15 B4WarmED). Therefore, this paper HWRC Ambient 10.5 ± 0.08 11.0 ± 0.07 24.0 ± 0.58 will only present results for three treat- HWRC +1.7° C 12.1 ± 0.05 12.9 ± 0.10 21.5 ± 0.63 ments: control (either control plots de- ° ± ± ± HWRC +3.4 C 13.7 0.06 14.3 0.07 20.1 0.51 pending on the presence of the studied Notes: The diff erences between the percentages of soil moisture are not part of the treatment but a result of it. species within each plot), +1.7 ° C and OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1613

+3.4 ° C. Mean aboveground and soil temperature along with soil Th e two herbaceous species studied are clonal, which means moisture at both sites for 2011 and 2012 are presented in Table 1 . that in theory the area occupied by clones may exceed the surface For photosynthesis, respiration and plant nutrient concentration, of the plot. In such a case, some ramets of a clone may be subjected data were only collected in the control and +3.4° C plots, due to to the treatment while others are not because they grow outside the the time required to complete photosynthesis and respiration plot. However, all were cut when the cables were in- measurements. Each treatment is repeated twice within a block, stalled, thus isolating ramets inside the plot from those outside. which is why the design is said to be “generalized”, and there are Th ree years later, a number of ramets have emerged nearby the three blocks per site for a total of 24 or 36 plots depending on the plots, but the slow growth of these two species (Meier et al., 1995) data type (2 sites × 2 or 3 treatments × 3 blocks × 2 within-block allows us to assume that this number is limited compared with the repetitions). number of ramets within the plots. In addition, the warming eff ect Th e plots are circular with an area of 7 m 2. In 2008, 11 bare-root extends to a 30-cm strip around the plots (unpublished data, seedlings of 11 woody species (121 plants per plot) were planted B4WarmED). Th us, the warming treatment may be mitigated to and 60 per species per plot were seeded in the fi rst year and 40 some extent by the presence of ramets outside the plots and the in the second. Th e warming treatment was turned on in spring exchanges that may occur between these and the ramets within the 2009; the growing seasons 2011 and 2012, when data for this proj- plot, but we expect this impact to be limited. ect were collected, were the third and fourth year of the warming treatment. Th e two studied species were naturally present in the Light and water availability — Each site is equipped with photometers plots and were chosen because they were present at both study sites that record data at regular intervals. To assess the degree of competi- and in the vast majority of plots. Th e mature trees in the stands are tion for light, measurements of photosynthetically active radiation not experimentally heated except for perhaps a small fraction of were taken directly above the leaves of E. macrophylla and M. ca- roots extending into warmed plots. nadense with a portable photometer (LI-189 with LI-190 sensor; Li- Cor, Lincoln, Nebraska, USA) in 2011. Measurements were taken on fi ve ramets per species per plot under diff use light conditions, that is to say, a cloudy day. Th e light readings were taken twice a week before and during canopy closure, and about once every 2 weeks aft er. Th e measurements taken at the plant level were then compared with the value re- corded by the photometer of the open habitat to calculate the percentage of available light that reached the plants. A mathematical model, the inverted Gom- pertz curve (Seber and Wild, 1989), was then applied on the data to establish a relationship between the percentage of available light and date throughout the season. It was then possible to calculate the proportion of available light at the plot level at any time during the season. Soil water content was measured con- tinuously in each plot using 30 cm long TDR probes (MiniTrase 6050 × 3; Soil Moisture Equipment Corp., Goleta, Cal- ifornia, USA) placed at 45° in the soil at a depth of 21 cm.

Total leaf area, reproductive output, and phenology— Various sizes of leaves of M. canadense and E. macrophylla were collected outside the plots. Th ese leaves were immediately scanned and their length, width, and area were estimated with the ImageJ soft ware (National Insti- FIGURE 2 Percentage of the total available light that reached plants of (A, C) Maianthemum canadense tutes of Health, Bethesda, Maryland, and (B, D) Eurybia macrophylla throughout the season for three warming treatments at the two study USA). We then determined that the best sites (CFC and HWRC). The curves were produced by fi tting an inverted Gompertz curve on the mean equation to estimate the area of these value for each treatment at each date. The arrows represent the average date of leaf unfolding (not leaves is a second order polynomial emergence) of the three treatments. regression (Sigma Plot for Windows 1614 • AMERICAN JOURNAL OF BOTANY

version 11.0; Systat, San Jose, California, USA). For M. canadense, this curve allowed us to determine that an irradiance of 800 μmol·m −2 ·s−1 relationship has been established from 69 leaves and corresponds to the of photons was suffi cient to saturate photosynthesis. All measure- × × 2 −1 following equation: Area = −1.002 + 1.613 width + 0.722 width ments were performed at a CO 2 concentration of 400 μmol·mol . (R 2 = 0.982), whereas for E. macrophylla, the equation was obtained Asat and Rn were measured on one diff erent individual per plot each from 53 leaves: Area = −2.395 + 8.040 × width + 0.859 × width 2 ( R 2 = time, within control and +3.4° C treatment plots. During measure- 0.987). Total leaf area per plant was estimated from measurements of ments, the LI-6400 console was located outside the plots; thus, the air individual leaf width and used to compare the size of plants among pumped by the instrument was at the control temperature, that is, the treatments. Th ese measures were taken once leaf unfolding was com- ambient temperature that was prevailing at that moment. Th us, leaf pleted in 2011, on 20 ramets per species per plot, or for all individuals temperatures during gas exchange measurements fl uctuated accord- in the plot if there were fewer than 20. Th e percentage of M. canadense ing to ambient temperature (ranged from 15.5° to 31.8° C; Appendix ramets that had fl owers was evaluated on one quarter of the plot, S1, see Supplemental Data with the online version of this article) but whereas the entire area of the plot was used for E. macrophylla. Flowers, were not diff erent between control and heated plots. Th erefore, the or infl orescences in the case of aster, were counted on each fl owering rates should be considered as having been measured at a standard individual, within both species. Th e fruits were harvested, counted, temperature, rather than at contrasting treatment temperatures. Th e dried, and weighed. Given the sequential fl owering and the trade-off measured leaves were then collected, scanned, dried, and weighed to between protecting the seeds against the wind and allowing open ac- calculate their specifi c leaf area (cm2 ·g−1 ). No leaves of M. canadense cess to pollinating insects, not all of the seeds of E. macrophylla could were collected at the HWRC site because they were too scarce. be collected. Plant cover per species has been estimated once a year since 2008, on two subplots of 1 m2 per plot. Annual changes in Leaf analysis —Leaves collected during the fi rst, fourth and sixth plant cover within each plot (2011/2009), for the two species under Asat measuring dates were sent to the Laboratoire Daishowa, Pavil- study was used to estimate the impact of global warming on clonal lon de l’Envirotron, Université Laval to assay for nutrient concentra- growth aft er taking into account warming impact of individual tions. Th ese leaves were dried at 60 °C for a minimum of 72 h. Leaf plant growth. Th e phenological stage was evaluated every week dur- material was then digested in H2 SO4 –H2 O2 –H2 SeO3 . N and P con- ing the growing season in 2011 and 2012. Stages used for analysis centrations were determined by colorimetry (Nkonge and Ballance, are leaf unfolding (date at which the fi rst leaves started to unfold) 1982); K, Ca, and Mg concentrations were determined by atomic ab- and leaf withering (date at which the leaves started to turn yellow) sorption spectroscopy. At mid-July, two leaves per plot per species to assess the duration of the photosynthetically active season. were collected for an estimate of chlorophyll concentration. Chloro- phyll was extracted in acetone and quantifi ed by spectrophotometry Photosynthesis and respiration — Photosynthetic and respiratory with a Spectro Cary 300 Bio (Agilent, Mississauga, , Canada), rates were measured using the portable gas exchange system LI-6400 and calculations were made according to Porra et al. (1989 ). (Li-Cor) during the 2011 growing season. Photosynthetic rates under saturating conditions (Asat) and night respiratory rates (Rn) were re- Statistical analyses —Analysis of variance with mixed eff ects on corded six times during the season, once plants had reached their a generalized split plot design was performed for each variable maximum size, i.e., from mid-June to late August. A light saturation recorded once during the growth season: total leaf area, data on

TABLE 2. Repeated measure analysis of variance with mixed eff ects testing the warming treatments and site eff ects on the date of leaf unfolding, leaf withering, and length of the growth season of Mainthemum canadense and Eurybia macrophylla and on mean air and soil temperature and soil moisture content. Site × Site × Treat × Site Treatment Treatment Year Site × Year Treat × Year Year Variable F P F P F P F P F P F P F P M. canadense Leaf unfolding1 0.96 0.38 22.4<0.001 0.07 0.93 59.1<0.001 0.01 0.92 3.950.03 1.32 0.29 Leaf wilting2 0.17 0.70 7.830.002 0.94 0.41 23.2<0.001 0.70 0.41 5.100.01 1.32 0.29 Length of season3 0.42 0.55 4.880.02 1.59 0.22 0.08 0.79 0.25 0.62 3.460.05 7.24 0.005 E. macrophylla Leaf unfolding4 55.80.002 40.7 <0.001 1.00 0.38 159<0.001 22.6 <0.001 1.44 0.25 2.64 0.09 Leaf wilting5 0.54 0.5 9.85<0.001 2.69 0.09 14.5<0.001 7.39 0.01 1.04 0.37 0.55 0.58 Length of season6 9.28 0.04 31.9 <0.001 4.600.02 2.84 0.10 28.3<0.001 2.19 0.13 2.75 0.08 Climate conditions Air temperature7 149<0.001 2694<0.001 1.86 0.17 2656<0.001 286<0.001 30.6<0.001 1.05 0.36 Soil temperature 8 77.9<0.001 1799<0.001 0.10 0.90 1492<0.001 110<0.001 2.44 0.10 6.76 0.002 Soil moisture content 9 13.9 0.02 7.39<0.001 0.41 0.67 9.680.003 5.79 0.02 0.26 0.78 0.30 0.75

Notes: Results of contrasts comparing treatments within year, the two sites pooled. 1 2011: Control a; +1.7° C b; +3.4° C b; 2012: Control a; +1.7° C b; +3.4° C c. 2 2011: Control a; +1.7° C a; +3.4° C a; 2012: Control a; +1.7° C a; +3.4° C b. 3 2011: Control b; +1.7° C a; +3.4° C a; 2012: Control ab; +1.7° C a; +3.4° C b. 4 2011: Control a; +1.7° C b; +3.4° C b; 2012: Control a; +1.7° C b; +3.4° C c. 5 2011: Control b; +1.7° C a; +3.4° C a; 2012: Control b; +1.7° C a; +3.4° C a. 6 2011: Control b; +1.7° C a; +3.4° C a; 2012: Control b; +1.7° C a; +3.4° C a. 7 2011: Control a; +1.7° C b; +3.4° C c; 2012: Control a; +1.7° C b; +3.4° C c. 8 2011: Control a; +1.7° C b; +3.4° C c; 2012: Control a; +1.7° C b; +3.4° C c. 9 2011: Control a; +1.7° C ab; +3.4° C b; 2012: Control a; +1.7° C b; +3.4° C c. OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1615

reproductive eff ort, chlorophyll concentrations, percentage cover, with mixed eff ects in which time was included as a categorical fi xed and light (the parameters of the Gompertz curves were compared eff ect. Th e Akaike information criterion was used to determine the between treatments: they are unique values obtained from repeated structure of dependence that best fi t the data. When the ANOVA measurements). Th e fi xed eff ect was the site in the main plot and indicated that the treatment was signifi cant, a protected LSD test was the treatment in the subplot. Th e random eff ects were block nested performed to determine which treatments diff ered. When the inter- in site and plot nested in [treatment × block(site)], in accordance action treatment × time (or year) was signifi cant, contrasts within with the experimental design. When the ANOVA indicated that each time (or year) were analyzed. Data were transformed when nec- the treatment was signifi cant, a protected least signifi cant diff er- essary. All analyses were performed using the mixed procedure in ence (LSD) test was performed to determine which treatments dif- SAS version 9.2 TS Level 1M0 (SAS Institute, Cary, North Carolina, fered. When the interaction treatment × site was signifi cant, USA). Th e only exception was percentage fl owering, which was ana- contrasts within each site were analyzed. lyzed with the GLIMMIX procedure to take into account the binary For measurements that were taken several times during the sum- nature of the response variable. To fi t a curve to the data for light mer, such as Asat, Rn, and leaf data (SLA and nutrients), or in 2 years availability, the nlin procedure was used. When data were collected at as for the phenology and climate data (stages or parameters were a single site only (e.g., M. canadense fl owering), a generalized ran- analyzed separately), we performed a repeated split-plot ANOVA dom block design was used.

RESULTS

Light and water availability — Th e light transmitted to the understory was maximal early in the season, before leaf emergence of trees and shrubs. During this period, the CFC site re- ceived about twice the light recorded at the HWRC site (Fig. 2). In the summer, when the total canopy had reached its seasonal maximum, E. macrophylla received less light under the two heated treatments than in the control plots, at both sites (P = 0.03; Appendix S2, see online Supplemental Data). Th e trend was the same for M. canadense (P = 0.06). Both air and soil temperatures differed among treat- ments as expected (Tables 1 and 2). Mean air temperature during the 2011 growing season was 0.1° C lower and 0.4 ° C higher than the mean of the previous 10 yr at CFC and HWRC, whereas in 2012, air temperature in the two locations diff ered from the mean by 0.2° C and −0.6° C, respectively. Therefore, the +1.7° C treatment in- duced conditions that were warmer than the typical conditions for the area, and even slightly warmer than the warmest year of the previous 10 yr. Th e water content of the soil was al- ways lower in the heated plots than in the control plots, and the +3.4° C plots were drier than the +1.7° C ( Tables 1 and 2 ).

Plant phenology — For M. canadense , a higher growth temperature advanced the onset of leaf unfolding in both FIGURE 3 Eff ects of the warming treatments on the mean (± SE) dates of leaf unfolding, leaf withering, years ( Table 2 , Fig. 3 ). Senescence and length of the growing season for (A) Maianthemum canadense and (B) Eurybia macrophylla at each started at the same time for all treat- experimental site. ments in 2011, but was earlier in the 1616 • AMERICAN JOURNAL OF BOTANY

+3.4 ° C compared with the +1.7° C and the control plots in 2012. fl owering ramet in the +1.7° C plots and 332 ± 31 seeds per fl ower- Th e early emergence in the heated plots allowed a longer growing ing ramet in the +3.4° C in plots. Th e dry mass of E. macrophylla season for M. canadense in 2011 than in the control plots. In 2012, seeds did not vary with growth temperature. the +1.7° C plots experienced a longer growing season than the Change in percentage cover across years can be used as a proxy +3.4 °C plots did, but the two heated treatments did not diff er in for clonal growth over time in both species, considering that plant terms of length of the growing season from the control plots. In the size was not aff ected by warming, except for E. macrophylla in one case of E. macrophylla , the time of leaf unfolding was advanced, and site. Cover remained stable in the control plots over the 3-year pe- leaf senescence was delayed by the higher growth temperatures in riod, whereas in many heated plots, the cover of both species both 2011 and 2012, resulting in a longer season for the plants of increased dramatically (online Appendix S3). However, plant re- the heated plots in both years. A much earlier warm-up in spring sponses to warming were variable across plots, such that there was 2012 than 2011 resulted in earlier leaf-out for both species in all not a statistical diff erence among treatments in change in cover treatments. from 2009 to 2011 ( Table 3 ; Appendix S3).

Specifi c leaf area— Specifi c leaf area (SLA) of M. canadense in- Total leaf area — Th e total leaf area of M. canadense was not af- creased during the growing season while it decreased for E. macro- fected by growth temperature, and individuals were smaller at the phylla ( Fig. 7A ). Growth temperature did not signifi cantly aff ect HWRC site than at the CFC site (Table 3, Fig. 4). Total leaf area for the SLA of either species ( Table 4 ). For M. canadense , there was a E. macrophylla was aff ected by growth temperature, but at one site nonsignifi cant trend for a higher SLA in plants of the +3.4° C plots . only (CFC), where control plants were smaller than plants in heated For E. macrophylla, the eff ect of growth temperature on the SLA plots. Th ere was a nonlinear response in which growth was greatest was almost signifi cant (P = 0.065); leaves in the control plots tended at the +1.7 °C treatment in both species, except for M. canadense at to have a higher SLA than the ones in the +3.4° C plots, at the CFC HWRC. site ( Fig. 7B ).

Reproductive output and clonal growth — Maianthemum ca- nadense only fl owered at the CFC site in accordance with their Photosynthesis and respiration— Photosynthetic rates under satu- larger size than at HWRC (Fig. 4). Warming had no signifi cant ef- rating light conditions (Asat) and respiratory rates measured at −2 −1 fect on the percentage of ramets that fl owered, the number of fl ow- night (Rn) were expressed on an area basis (μmol CO2 ·m ·s ) and −1 −1 ers per ramet, nor the number of fruits per ramet (Table 3, Fig. 5). on a mass basis (nmol CO2 ·g ·s ) to take into account the trends However, the dry mass per was higher in plants from the observed for the specifi c leaf area. Asat of M. canadense decreased +1.7 ° C plots than in both control and +3.4° C plots. with time ( Table 4 ). When expressed on an area-based unit, Asat Eurybia macrophylla only fl owered in the heated plots and did tended to be higher at the ambient temperature than at +3.4° C: the so at both sites. Th ree times more plants were in fl ower in the diff erences were signifi cant for a few dates only, mostly at the CFC +1.7 ° C plots than in the +3.4° C in plots ( Table 3 , Fig. 6 ). Th e fl ow- site ( Fig. 8A, B ). Th e diff erences between treatments were less obvi- ering ramets in the +1.7° C plots had almost twice as many infl ores- ous when Asat was expressed on a mass basis (only one date for cences as those in the +3.4° C plots. Th ese two variables presented which control plants had higher Asat than the +3.4° C; data avail- a nonlinear response in both species. Although we could not collect able only for the CFC site, Fig. 8E). Leaves from control plots tended all seeds produced, we counted on average 355 ± 23 seeds per to have a lower SLA ( Fig. 7A ), which could explain the larger diff er- ences between treatments observed for TABLE 3. Analysis of variance with mixed eff ects to test warming treatments and site eff ects on the total leaf area-based than for mass-based Asat. area, fl ower and fruit production, percentage plant cover, and leaf chlorophyll concentration of Mainthemum For E. macrophylla , Asat per leaf canadense and Eurybia macrophylla . area were diff erent among treatments at the CFC site (Asat, > Asat, ), Site Treatment Site × Treatment +3.4 ° C control but not at HWRC ( Table 4 ; Fig. 8C, D ). Variable F P F P F P Th e diff erence between treatments dis- M. canadense appeared when Asat was expressed on Total leaf area 29.14 0.01 2.44 0.11 1.39 0.27 a per mass basis (Fig. 8F, G). Th e dif- Flowering (%) — — 1.38 0.29 — — Number of fl owers / ramet — — 0.07 0.94 — ference caused by the change in units Number of fruits / ramet — — 2.37 0.13 — — might be due to the specifi c leaf area Dry mass per fruit (mg) — — 6.500.01 ——that tended to be higher in plants Percent cover 2011/2009 2.02 0.23 2.61 0.09 1.05 0.37 growing at the ambient temperature at Total chlorophyll (mg·g−1 ) — — 0.07 0.94 — — the CFC site. Asat did not diff er be- Chlorophyll a/b — — 0.38 0.69 — — tween treatments either on an area- E. macrophylla based or on a mass-based unit for the Total leaf area 13.98 0.02 6.270.01 4.890.02 Flowering (%) 0.81 0.42 27.59 <0.001 2.7 0.06 plants of the HWRC sites, in accor- Number of fl owers / ramet 5.38 0.08 8.060.01 0.82 0.37 dance with the very similar SLA re- Number of fruits / ramet — — — — — — ported for the two treatments at that Dry mass per fruit (mg) 0.19 0.68 0.99 0.37 2.32 0.19 site ( Fig. 7C ). Percent cover 2011/2009 3.99 0.12 1.77 0.19 2.11 0.14 For both species, Rn was not strongly −1 Total chlorophyll (mg·g ) 0.65 0.47 1.18 0.32 2.45 0.11 affected by the warming treatment Chlorophyll a/b 0.06 0.82 2.45 0.11 0.26 0.77 (Table 4; Fig. 8H–K). Rn tended to be OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1617

Appendix S5). Th ere was no eff ect of the treatments on the total chlorophyll concentration (chl a +b) or on the ratio of chlorophyll a to b ( Table 3 ) in both species.

DISCUSSION

In general, experimental warming in- fl uenced some but not all measures of phenology, growth, metabolism, and chemistry for the two forest herbaceous species studied, M. canadense and E. macrophylla , yet, aft er three growing seasons, these translated into increased reproductive output. Th e main impact of the warming treatments appears to be the earlier emergence of the under- story herbs, which most likely in- creased their C gain during the high light period in spring before canopy closure. Many variables responded to warming in a nonlinear fashion, for instance, leaf area and some of the re- productive output variables. Positive warming eff ects might thus be limited to a narrow window of temperature increases. Th ese nonlinear trends also remind us that predicting the response of plants is complex: Multiple factors are infl uenced by warming and are thus involved in the response of plants in natural habitats ( Fig. 1 ); this limits our capacity to predict how specifi c species will respond to warming without em- FIGURE 4 Eff ect of the warming treatments on the mean (± SE) total leaf area of (A) Mainthemum ca- pirical data. nadense and (B) Eurybia macrophylla at study sites CFC and HWRC. Letters represent signifi cant diff er- Maianthemum canadense and E. ences according to protected LSD tests. macrophylla emerged earlier in the heated plots, and they likely fixed a large amount of their annual C during higher in control than in +3.4° C plots but diff erences were signifi - the open canopy period. Spring photosynthesis is a key element for cant for only a few dates in both species. Rn expressed on per leaf the productivity of many understory species of hardwood forests area and on a per mass unit exhibited very similar changes over ( Bazzaz and Bliss, 1971 ; Brown et al., 1985 ; Lapointe, 2001 ; Neufeld time and between treatments (Fig. 8L–N ). and Young, 2014 ), although many understory species do emerge during or after canopy closure. Photosynthetic rates of spring Leaf nutrients and chlorophyll — Time aff ected most of the leaf nu- emerging species are at the highest while the light is at its maximum trient concentrations in both species, whereas growth temperature availability, and rates drop aft er canopy closure. For some of these had limited eff ect ( Table 5 ). Th e nutrient data were analyzed both species, photosynthetic rates drop aft er only a few weeks, some- on a mass and on a leaf area based unit. In E. macrophylla , some times even days ( Larcher, 2003 ). Th is strategy is called the “pheno- nutrient concentrations (mg·g−1 ) increased with time (Ca and Mg) logical escape” because these species advance their emergence to while others decreased (N, P, and K). Ca and Mg concentrations avoid the shade of taller species that develop later. However, early were higher in leaves of plants grown in the heated plots early in the emergence also increases the risk of spring frost, which could season, but this diff erence was no longer present at the second and strongly reduce annual C gain. third harvests (Appendix S4). In M. canadense, K, Ca, and Mg Aft er tree leaf emergence, plants in the heated plots received less concentrations increased with time on a mass basis, whereas the light due to the improved growth of the transplanted tree seedlings pattern was not clear for N and P (Appendix S5). P concentration in these plots than in the controls. However, reduced light during on an area basis was higher in plants grown in the heated plots at the summer did not aff ect SLA or the chlorophyll a /b ratio, two fi rst harvest, whereas K concentrations on a mass basis was higher variables known to be sensitive to light availability. Th ese results in plants of the control plots but only at the second harvest ( Table 5 ; indicate that the lowest light levels that prevail in the heated plots in 1618 • AMERICAN JOURNAL OF BOTANY

therefore important to quantify the warming impact on the diff erent func- tional groups within a forest, including the overstory trees, to determine which understory plants if any will be able to take advantage of earlier leafi ng under a warmer climate. It is expected that the effect of warming on the pheno- logical escape strategy used by many understory species will most likely vary with composition and successional stage of the forest stand. Furthermore, results from experimental warming studies will need to be confi rmed by observational data recorded along time series because experimental warming may underpredict the advances in spring phenology ( Wolkovich et al., 2012 ). Not only emergence, but also senes- cence of the two herbaceous species was affected by the warming treat- ment. Maianthemum canadense se- nesced earlier in warmed plots in 2012, similarly to what has been reported for another spring fl owering species, Smi- lacina japonica ( Ishioka et al., 2013 ), or for heated plots containing mostly M. canadense and Uvularia sessilifolia ( Farnsworth et al., 1995 ). In contrast, E. macrophylla plants lasted longer in the heated plots in both years, whereas other studies reported either no change FIGURE 5 Eff ect of the warming treatments on diff erent aspects of the reproductive output of Maianthe- in three forest summer herbs (Ishioka mum canadense at the CFC site. (A) The mean (± SE) percentage of ramets that fl owered per treatment. (B) et al., 2013 ) or even an earlier senes- The mean (± SE) number of fl owers per ramet. (C) The mean (± SE) number of fruits per ramet. (D) The cence in Panax quinquefolius ( Jochum mean ( ± SE) dry mass of individual fruits. Letters represent diff erences according to a protected LSD test. et al., 2007 ). Th ere are numerous factors controlling the initiation of senescence, among them environmental stresses, summer are not dramatic enough to induce strong morphological photoperiod, temperature ( Munné-Bosch and Alegre, 2004 ), or or physiological acclimation, compared with the change in light source–sink relationships (Lapointe, 2001). Th e diff erent timing in from spring to summer that induce a reduction in Asat in many the senescence of the two species can be partially explained by their species (Taylor and Pearcy, 1976). It is also possible that these reproductive strategies: early bloomer for M. canadense and late plants were already as much acclimated to shade as their plasticity summer bloomer for E. macrophylla . Th e fruits of M. canadense are allowed them. In this experimental setup, the overstory canopy is ripe in September, and senescence seems to be induced once seeds not exposed to the treatment. However, the saplings of woody spe- complete their development. Th ere is no other major sink within cies planted in the plots were heated. Th ese saplings also advanced the plant at ripening as carbohydrate storage in rhizomes have their phenology in response to warming (Schwartzberg et al., 2014; already taken place (L. Lapointe, unpublished data). Eurybia macro- and data not published, B4WarmED) as reported in other studies phylla seeds mature until the very end of the season; some seeds ( Fu et al., 2014 ; Kaye and Wagner, 2014 ). While herbaceous species were actually not ripe when the plant fi nally senesced. Th is species rely more strictly on the temperature and snow melt as a signal for appears to lengthen its growth season as much as possible, and emerging ( Farnsworth et al., 1995 ; Iversen et al., 2009 ; De Frenne night frost most likely induces senescence. Delayed senescence un- et al., 2011 ; Cornelius et al., 2013), the response of trees to tempera- der a warmer climate would allow the plant to mature more seeds. ture is more complex and varies among species because of diff er- Th e eff ect of warming on shoot senescence might therefore depend ent sensitivities to temperature, photoperiod, and winter chilling on the reproductive strategy of the species, which may partly ex- (Murray et al., 1989; Borchert et al., 2005; Vitasse et al., 2009; plain the diversity of responses reported in the literature ( Menzel Körner and Basler, 2010). Although most warming studies have et al., 2006 ). used seedlings or saplings rather than mature trees for practical The effect of the heating treatment on Asat and Rn was not reasons, ontological diff erences cause young trees to leaf out earlier pronounced in either species, as reported for other understory than their mature conspecifi cs, even aft er taking into account verti- herbaceous species in soil-warming alone experiments (Ishioka cal diff erences in temperature within the forest (Vitasse, 2013). It is et al., 2013). The results support the hypothesis that there was a OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1619

temperature response curve (Lambers et al., 1998). However, warming stud- ies in general indicate limited impacts on leaf N, about 3.3% increase ac- cording to a meta-analysis (Bai et al., 2013 ). In the current study, the effect of warming on the nutrient content of the leaves was also very small. Th ere- fore, the main impact of the warming treatment appears to be on the dura- tion of the photosynthetic period rather than on instantaneous gas exchange rates. The modest response of Asat to warming of E. macrophylla and M. canadense is also consistent with the location of the experimental site rela- tive to their north–south range dis- tributions. For the 11 B4WarmED tree species, those with northern ranges close to the study site (e.g., maples, oaks) had increased Asat in response to warming, whereas those whose southern range was close to the study sites (fi r, ) had decreased Asat ( Reich et al., 2015 ). Th e ranges of both E. macrophylla and M. canadense ex- tend far to the north and the south of northern Minnesota; thus, perhaps both the ambient and warmed thermal regimes fall well within the general range of variability to which the spe- cies are adapted. FIGURE 6 Effect of the warming treatments on different aspects of the reproductive output of Eury- Th e increased reproductive output bia macrophylla; with results shown pooled across both sites. (A) The mean (± SE) percentage of ra- reported in both species supports the mets that fl owered per treatment. (B) The mean ( ± SE) number of infl orescences per ramet. (C) The mean assessment that plants in heated plots ( ±SE) dry mass of individual seeds (akenes). Letters represent differences according to protected fi xed more C during the season, most LSD tests. likely due to their extended growth season. Indeed, it is remarkable that E. macrophylla only fl owered and pro- shift of the thermal optimum of Asat and Rn, with no change in duced seeds in the heated plots despite no signifi cant change in the elevation of the curve, as the rates in the heated plots were Asat or total leaf area. Small but cumulative diff erences in total C lower when measured at the common ambient temperature. For gain over the season could translate, 3 years later, into a signifi cant the planted deciduous tree species in B4WarmED, acclimation investment into sexual reproduction in heated plots. In another of Asat and Rn was a general response (Sendall et al., 2015; spring fl owering understory species, Anemone nemorosa, an early P. Reich, unpublished data). Although it was not possible to es- emergence was correlated with an increased percentage of fl ower- tablish the temperature response curves of Asat in the field, the ing and increased seed mass ( De Frenne et al., 2011 ). However, in

literature indicates that most C3 plants have a broad optimum summer fl owering species, warming did not improve total seed ( Sage and Kubien, 2007 ), located between 15° and 30° C ( Larcher, production (De Frenne et al., 2009), contrary to what we reported 2003), as was the case for the deciduous trees in B4WarmED here for E. macrophylla. Th e response of reproductive output to ( Sendall et al., 2015 ). Moreover, Asat of the juvenile trees was warming appears to be partly dependent upon their phenology as well matched to their thermal regimes in the warmed plots (De Frenne et al., 2009, 2011 ), but the impact of warming on plant as in the ambient plots in 2009 to 2011 (Sendall et al., 2015). If phenology needs to be characterized for many species before we true for the two herb species, a broad temperature optimum can generalize the response of understory species to climate would also contribute to minimal differences in Asat across warming. treatments. The nonlinear response of the reproductive output of both Additionally, other aspects of plant function can alter realized species, which was also observed for the total leaf area and the carbon exchange rates, beyond the shape of the response curve. percentage cover (although both not significant), might be re- For example, changes in leaf N induced by warming could trans- lated to potential water stress at the higher temperature regime. late into changes in elevation of Asat and Rn anywhere along a Soil water content decreased with the increase in temperature, 1620 • AMERICAN JOURNAL OF BOTANY

the control plots (data not shown). The reduced growth recorded in 2011 at the highest warming treatment compared with the milder warming treatment could be the result of cu- mulative effects of water stress from the two previous seasons ( Inghe and Carl, 1985 ; Inghe and Tamm, 1988 ), which were both relatively dry in this area ( Reich et al., 2015 ). We noted the presence of a patho- gen on E. macrophylla leaves at the CFC site. This pathogen is the needle rust, Coleosporium asterum (Dietel) Syd. & P. Syd, of which E. mac- rophylla is an alternate host. Only E. macrophylla in the control plots were attacked by the pine needle rust, which could explain why these plants were smaller and tended to have a higher SLA than plants in heated plots. Th e urediospores of C. asterum were cer- tainly on E. macrophylla leaves in each plot, but conditions suitable for germi- nation were only found in the control plots. Th e optimal temperature for ure- diospore germination is 20° C, while only 1% of the urediospores germi- nate at 30° C and none at 35° C (Fergus, 1959 ; Nicholls et al., 1967). Th e heated plots were most likely too warm for germination, but perhaps also too dry. We observed on a regular basis throughout the season that the dew FIGURE 7 Eff ect of the warming treatments on the mean (± SE) specifi c leaf area of Maianthemum had already evaporated in the heated canadense at (A) the CFC site and of Eurybia macrophylla at (B) CFC and (C) HWRC site throughout plots as we arrived at the study site in the season. There was no M. canadense collected at HWRC site because there were too few plants in the morning but was still present on the plots. the plants of control plots. In a warmer future, changes in the phenology, se- and this effect was present throughout the season ( Rich et al., verity, and distribution of biotic agents are to be expected, with 2015 ). However, the summer of 2011 was not dry enough to reduce potentially complex impacts on plant growth and reproduction stomatal conductance in the +3.4 ° C treatment compared with (e.g., Liu et al., 2011 ).

TABLE 4. Analysis of variance with mixed eff ects testing the warming treatments, site, and time eff ects on the specifi c leaf area and on the Asat and Rn both on an area-based and on a mass-based units for Maianthemum canadense and Eurybia macrophylla . Site × Treatment × Site × Treat × Site Treatment Treatment Time Site × Time Time Time VariableF P F P F P F P F P F P F P M. canadense SLA (cm2 ·g−1 ) — — 3.11 0.22 — — 5.37<0.001 — — 0.95 0.45 — — Asat (μmol·m−2 ·s−1 ) 2.05 0.23 10.200.01 1.22 0.29 18.67 <0.001 1.81 0.12 2.420.04 2.770.02 Asat (nmol·g−1 ·s−1 ) — — 6.270.03 — — 2.72 0.03 — — 1.64 0.17 — — Rn (μmol·m−2 ·s−1 ) 1.37 0.31 3.54 0.08 4.630.05 9.53 <0.001 15.420.001 1.15 0.34 1.88 0.11 Rn (nmol·g−1 ·s−1 ) — — 18.840.003 — — 9.09 <0.001 — — 2.540.04 —— E. macrophylla SLA (cm2 ·g−1 ) 0.16 0.71 6.39 0.06 3.81 0.12 7.46<0.001 0.69 0.64 1.67 0.15 0.38 0.86 Asat (μmol·m−2 ·s−1 ) 0.84 0.41 5.510.03 7.36 0.01 1.16 0.33 1.08 0.38 0.96 0.45 0.27 0.93 Asat (nmol·g−1 ·s−1 ) 0.46 0.54 0.01 0.93 0.75 0.40 6.17<0.001 1.43 0.22 1.62 0.16 0.18 0.97 Rn (μmol·m−2 ·s−1 ) 3.62 0.13 5.140.04 0.46 0.51 35.32 <0.001 11.37<0.001 0.42 0.84 1.23 0.30 Rn (nmol·g−1 ·s−1 ) 1.40 0.30 9.160.01 0.34 0.57 35.70 <0.001 7.80<0.001 1.21 0.31 1.02 0.41 OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1621

FIGURE 8 Eff ect of the warming treatments for Maianthemum canadense and Eurybia macrophylla through the season. (A–G) On mean (± SE) photosyn- thetic rates under saturating light conditions (Asat) reported on (A–D) an area basis and (E–G) a mass basis. (H–N) On mean ( ±SE) respiratory rates measured at night (Rn) reported on (H–K) an area basis and (L–N) a mass basis. Asterisks represent signifi cant diff erences per date based on contrasts analyses done a posteriori when the interaction treatment × time or treatment × site × time was signifi cant.

CONCLUSION retarded in heated plots. The growing season was nevertheless longer, and although it did not translate into consistently larger Warming induced an earlier emergence in both M. canadense and plants, it did allow some individual plants to grow larger and pro- E. macrophylla, two early-emerging species, whereas the timing duce more fl owers and seeds. Midsummer conditions in warmed of senescence was inconsistent, being advanced, unaffected, or plots (lower light and soil water availability) might have counteracted 1622 • AMERICAN JOURNAL OF BOTANY

TABLE 5. Analysis of variance with mixed eff ects testing the warming treatments, site, and time eff ects on the N, P, K, Ca, and Mg concentration in leaves of Mainthemum canadense and Eurybia macrophylla . Site × Site × Treat × Site Treatment Treatment Time Site × Time Treat × Time Time Variable F P F P F P F P F P F P F P M. canadense N (g·m −2 ) — — 3.52 0.09 — — 13.81<0.001 — — 0.11 0.89 — — N (mg·g−1 ) — — 1.75 0.22 — — 1.29 0.30 — — 3.21 0.06 — — P (g·m−2 ) — — 8.110.02 — — 2.51 0.11 — — 0.77 0.48 — — P (mg·g−1 ) — — 1.82 0.21 — — 2.54 0.11 — — 0.24 0.79 — — K (g·m−2 ) — — 0.01 0.91 — — 5.360.01 — — 1.29 0.30 — — K (mg·g−1 ) — — 11.120.01 — — 4.810.02 — — 3.830.04 —— Ca (g·m−2 ) — — 0.12 0.74 — — 2.23 0.14 — — 2.90 0.08 — — Ca (mg·g−1 ) — — 3.81 0.08 — — 11.69 <0.001 — — 1.08 0.36 — — Mg (g·m−2 ) — — 1.29 0.28 — — 5.050.02 — — 0.44 0.65 — — Mg (mg·g−1 ) — — 1.38 0.27 — — 6.870.01 — — 0.13 0.88 — — E. macrophylla N (g·m−2 ) 1.91 0.24 0.66 0.43 0.71 0.41 2.47 0.10 0.75 0.48 0.85 0.43 2.72 0.08 N (mg·g−1 ) 0.38 0.57 2.79 0.11 0.25 0.63 33.23<0.001 3.740.03 6.06 0.01 0.21 0.81 P (g·m−2 ) 24.070.01 1.14 0.30 0.38 0.55 0.27 0.77 0.56 0.58 1.13 0.33 0.53 0.59 P (mg·g−1 ) 16.510.02 2.58 0.12 1.00 0.33 9.60<0.001 0.35 0.71 2.39 0.11 0.18 0.84 K (g·m−2 ) 30.500.01 0.00 0.97 0.01 0.94 1.37 0.27 1.69 0.20 0.27 0.77 0.39 0.68 K (mg·g−1 ) 20.240.01 0.35 0.71 1.61 0.23 12.87<0.001 1.14 0.33 0.36 0.84 0.25 0.86 Ca (g·m−2 ) 0.24 0.65 21.490.00 0.55 0.47 48.02<0.001 1.18 0.32 2.04 0.15 0.11 0.90 Ca (mg·g−1 ) 7.34 0.05 6.800.02 0.69 0.42 107.03<0.001 1.37 0.27 6.100.01 0.58 0.57 Mg (g·m−2 ) 2.23 0.21 7.480.01 2.72 0.11 28.27<0.001 0.46 0.64 5.570.01 0.64 0.54 Mg (mg·g−1 ) 18.160.01 8.41 0.01 1.83 0.19 39.06<0.001 0.56 0.58 12.26<0.001 0.17 0.84

the positive eff ects of the earlier emergence and thus explain the LITERATURE CITED limited impact of the lengthened growing season on the growth of M. canadense and E. macrophylla , as well as only modest and in- A r ft , A. M. , M. D. Walker , G. Gurevitch , J. M. Alatalo , M. S. Bret-Harte , M. Dale , M. Diemer , et al. 1999 . Responses of tundra plants to experimental consistent impacts on leaf gas exchange rates. Furthermore, leaf warming: Meta-analysis of the international tundra experiment. Ecological nutrient concentration was not generally increased by the warming Monographs 69 : 491 – 511 . treatment, which could partly explain the lack of positive re- Bai , E. , L. Shanlong , W. Xu , W. Li , W. Dai , and P. Jiang . 2013 . A meta-analysis sponse of photosynthesis and respiration to the warming treat- of experimental warming eff ects on terrestrial nitrogen pools and dynamics. ment. Th e earlier emergence and heightened reproduction would New Phytologist 199 : 441 – 451 . be advantageous under a warmer climate, especially if shrub and Bazzaz , F. A. , and L. C. Bliss . 1971 . Net primary production of herbs in a tree strata do not shift their emergence as much as the herbs. We central Illinois deciduous forest. Bulletin of the Torrey Botanical Club 9 8 : thus need to quantify the warming impact on the phenology not 90 – 94 . only for diff erent forest plant groups but also for specifi c species Borchert , R. , K. Robertson , M. D. Schwartz , and G. Williams-Linera . 2005 . to predict how diff erent forest ecosystems will respond to global Phenology of temperate trees in tropical climates. International Journal of warming. Biometeorology 50 : 57 – 65 . Brouillet , L. 2006 . Eurybia . In Flora of North America Editorial Committee [eds.], 1993+, Flora of North America North of Mexico, vol. 20, Asteraceae, ACKNOWLEDGEMENTS 365–375. Oxford University Press, New York, New York, USA. Brown , R. L. , J. W. Ashmun , and L. F. Pitelka . 1985 . Within-species and We thank Stefan Hupperts, Colton Miller, Hannah Tuntland, between-species variation in vegetative phenology in two forest herbs. Camdilla Wirth, Michelle Cummings, Kaleb Remski, Philip Green, Ecology 66 : 251 – 258 . Ryan Steenson, Byron Meeks, Jon Shepard, Kaleb Welch, Lindsay Campbell , C. , L. Atkinson , J. Zaragoza-Castells , M. Lundmark , O. Atkin , and Wallis, and Elliot Vaughan for field assistance and Dominique V. Hurry . 2007 . Acclimation of photosynthesis and respiration is asyn- Manny, Antoine Daignault, and Timothée Bitsch for laboratory chronous in response to changes in temperature regardless of plant func- assistance. We thank Roy Rich and Mikhail Titov for providing tional group. New Phytologist 176 : 375 – 389 . data, Gilbert Ethier and Steeve Pepin for scientific advice, and Castro , H. , and H. Freitas . 2009 . Above-ground biomass and productivity in anonymous reviewers for helpful comments and suggestions the Montado: From herbaceous to shrub dominated communities. Journal of Arid Environments 73 : 506 – 511 . during the preparation of this manuscript. This research was Cornelius , C. , N. Estrella , H. Franz , and A. Menzel . 2013 . Linking altitudinal funded by the Offi ce of Science (BER), U.S. Department of Energy, gradients and temperature responses of plant phenology in the Bavarian Grant No. DE-FG02-07ER64456, the Minnesota Department of Alps. Plant Biology 15 ( supplement 1 ): 57 – 69 . Natural Resources, the University of Minnesota College of Food, De Frenne, P. , J. Brunet , A. Shevtsova , A. Kolb , B. J. Graae , O. Chabrerie , Agricultural, and Natural Resources Sciences, the UM Wilderness S. A. O. Cousins , et al. 2011 . Temperature eff ects on forest herbs assessed Research Foundation, and NSERC and FRQNT post graduate by warming and transplant experiments along a latitudinal gradient. Global fellowship to M.H.J. Change Biology 17 : 3240 – 3253 . OCTOBER 2015 , VOLUME 102 • JACQUES ET AL. —RESPONSE OF TWO UNDERSTORY HERBS TO WARMING • 1623

De Frenne , P. , B. J. Graae , A. Kolb , J. Brunet , O. Chabrerie , S. A. O. Cousins , Körner , C. , and D. Basler . 2010 . Phenology under global warming. Science 327 : G. Decocq , et al. 2010 . Signifi cant eff ects of temperature on the reproduc- 1461 – 1462 . tive output of the forest herb Anemone nemorosa L. Forest Ecology and LaFrankie , J. V. 2003 . Maianthemum . In Flora of North America Editorial Management 259 : 809 – 817 . Committee [eds.]. 1993+. Flora of North America North of Mexico, vol. 33, De Frenne , P. , A. Kolb , K. Verheyen , J. Brunet , O. Chabrerie , G. Decocq , M. Liliaceae, 206–208. Oxford University Press, New York, New york, USA. Diekmann , et al. 2009 . Unravelling the eff ects of temperature, latitude and Lambers , H. , F. S. I. II Chapin , and T. L. Pons . 1998 . Plant physiological ecol- local environment on the reproduction of forest herbs. Global Ecology and ogy. Springer, New York, New York, USA. Biogeography 18 : 641 – 651 . Lapointe , L. 2001 . How phenology infl uences physiology in deciduous forest Dormann , C. F. , and S. J. Woodin . 2002 . Climate change in the Arctic: Using spring ephemerals. Physiologia Plantarum 113 : 151 – 157 . plant functional types in a meta-analysis of fi eld experiments. Functional Larcher , W. 2003 . Physiological plant ecology. Springer, Berlin, Germany. Ecology 16 : 4 – 17 . Lin , D. L. , J. Y. Xia , and S. Q. Wan . 2010 . Climate warming and biomass accu- Farnsworth , E. J. , J. Núñez-Farfán , S. A. Careaga , and F. A. Bazzaz . 1995 . mulation of terrestrial plants: A meta-analysis. New Phytologist 188 : 187 – 198 . Phenology and growth of three temperate forest life forms in response to Liu , Y. , P. B. Reich , G. Li , and S. Sun . 2011 . Shift ing phenology and abundance artifi cial soil warming. Journal of Ecology 83 : 967 – 977 . under experimental warming alters trophic relationships and plant repro- Fergus , C. L. 1959 . Th e infl uence of environment upon germination and ductive capacity. Ecology 92 : 1201 – 1207 . longevity of aeciospores and urediospores of Coleosporium solidaginis. Manzoni , S. , J. P. Schimel , and A. Porporato . 2012 . Responses of soil microbial Mycologia 51 : 44 – 48 . communities to water stress: Results from a meta-analysis. Ecology 93 : 930 – 938 . Fu , Y. S. H. , M. Campioli , Y. Vitasse , H. J. De Boeck , J. Van den Berge , H. Marie-Victorin , F. E. C. 1935 . Flore laurentienne. Imprimerie De-la-Salle, AbdElgawad , H. Asard , et al. 2014 . Variation in leaf fl ushing date infl uences Montreal, , Canada. autumnal senescence and next year’s fl ushing date in two temperate tree spe- Meier , A. J. , S. P. Bratton , and D. C. Duff y . 1995 . Possible ecological mecha- cies. Proceedings of the National Academy of Sciences, USA 111: 7355 – 7360 . nisms for loss of vernal-herb diversity in logged eastern deciduous forests. Ge , Q. , H. Wang , and J. Dai . 2015 . Phenological response to climate change in Ecological Applications 5 : 935 – 946 . China: A meta-analysis. Global Change Biology 21 : 265 – 274 . Menzel , A. , T. H. Sparks , N . Estrella , E . Koch , A . Aasa , R . Ahas , K . Alm-Kübler , Gilliam , F. S. 2007 . Th e ecological signifi cance of the herbaceous layer in tem- et al. 2006 . European phenological response to climate change matches the perate forest ecosystems. Bioscience 57 : 845 – 858 . warming pattern. Global Change Biology 12 : 1969 – 1976 . Gleason , H. A. , and A. R. T. H. Cronquist . 1963 . Manual of vascular plants of Munné-Bosch , S. , and L. Alegre . 2004 . Die and let live: Leaf senescence contributes northeastern United States and adjacent Canada. Van Nostrand, Princeton, to plant survival under drought stress. Functional Plant Biology 31 : 203 – 216 . New Jersey, USA. Murray , M. B. , M. G. R. Cannell , and R. I. Smith . 1989 . Date of budburst of Griffi n , D. M. 1981 . Water and microbial stress. Advances in Microbial Ecology 15 tree species in Britain following climatic warming. Journal of Applied 5 : 91 – 136 . Ecology 26 : 693 – 700 . Handler , S. , M. J. Duveneck , L. Iverson , E. Peters , R. M. Scheller , K. R. Wythers , Neufeld , H. S. , and R. D. Young . 2014 . Ecopysiology of the herbaceous layer in L. Brandt , et al. 2014 . Minnesota forest ecosystem vulnerability assessment temperate deciduous forests. In F. S. Gilliam [ed.], Th e herbaceous layer in and synthesis: A report from the Northwoods Climate Change Response forests of Eastern North America, 2nd ed., 35–95. Oxford University Press, Framework Project. General Technical Report NRS-XX. Northern Research Oxford, UK. Station. U.S. Department of Agriculture, Forest Service, Newtown Square, Nicholls , T. H. , R. F. Patton , and E. P. Van Arsdel . 1967 . Life cycle and seasonal Pennsylvania, USA. Available at http://www.fs.fed.us/nrs/pubs/gtr/gtr_ development of Coleosporium pine needle rust in Wisconsin. Phytopathology nrs133.pdf [accessed 14 July 2015]. 58 : 822 – 829 . Hovenden , M. J. , K. E. Wills , R. E. Chaplin , J. K. V. Schoor , A. L. Williams , Nkonge , C. , and G. M. Ballance . 1982 . A sensitive colorimetric procedure for

Y. Osanai , and P. C. D. Newton . 2008 . Warming and elevated CO2 aff ect nitrogen determination in micro-Kjeldahl digests. Journal of Agricultural the relationship between seed mass, germinability and seedling growth in and Food Chemistry 30 : 416 – 420 . Austrodanthonia caespitosa , a dominant Australian grass. Global Change Parmesan , C. 2007 . Infl uences of species, latitudes and methodologies on es- Biology 14 : 1633 – 1641 . timates of phenological response to global warming. Global Change Biology Inghe , O. , and O. T. Carl . 1985 . Survival and fl owering of perennial herbs. IV. 13 : 1860 – 1872 . Th e behaviour of Hepatica nobilis and Sanicula europaea on permanent Pearson , C. J. , and S. G. Shah . 1981 . Eff ects of temperature on seed production, plots during 1943–1981. Oikos 45 : 400 – 420 . seed quality and growth of Paspalum dilatatum. Journal of Applied Ecology Inghe , O. , and C. O. Tamm . 1988 . Survival and fl owering of perennial herbs. V. 18 : 897 – 905 . Patterns of fl owering. Oikos 51 : 203 – 219 . Porra , R. J. , W. A. Th ompson , and P. E. Kriedemann . 1989 . Determination IPCC . 2007 . Contribution of Working Groups I, II and III to the Fourth of accurate extinction coeffi cients and simultaneous equations for assaying Assessment Report of the Intergovernmental Panel on Climate Change. chlorophyll a and chlorophyll b extracted with four diff erent solvents— Intergovernmental Panel on Climate Change, Geneva, Switzerland. Verifi cation of the concentration of chlorophyll standards by atomic ab- Ishioka , R. , O. Muller , T. Hiura , and G. Kudo . 2013 . Responses of leafi ng sorption spectroscopy. Biochimica et Biophysica Acta 975 : 384 – 394 . phenology and photosynthesis to soil warming in forest-fl oor plants. Acta Reich , P. B. , K. M. Sendall , K. Rice , R. L. Rich , A. Stefanski , S. E. Hobbie , and Oecologica 51 : 34 – 41 . R. A. Montgomery . 2015 . Geographic range predicts photosynthetic and Iversen , M. , K. A. Brathen , N. G. Yoccoz , and R. A. Ims . 2009 . Predictors of growth response to warming in co-occurring tree species. Nature Climate plant phenology in a diverse high-latitude alpine landscape: Growth forms Change 5 : 148 – 152 . and topography. Journal of Vegetation Science 20 : 903 – 915 . Rich , R. L. , A. Stefanski , R. A. Montgomery , S. E. Hobbie , B. A. Kimball , and Jackson , R. B. , J. Canadell , J. R. Ehleringer , H. A. Mooney , O. E. Sala , and E. D. P. B. Reich . 2015 . Design and performance of combined infrared canopy Schulze . 1996 . A global analysis of root distributions for terrestrial biomes. and belowground warming in the B4WarmED (Boreal Forest Warming at Oecologia 108 : 389 – 411 . an Ecotone in Danger) experiment. Global Change Biology 21 : 2334 – 2348 . Jochum , G. M. , K. W. Mudge , and R. B. Th omas . 2007 . Elevated temperatures Routhier , M.-C. , and L. Lapointe . 2002 . Impact of tree leaf phenology on increase leaf senescence and root secondary metabolite concentrations in growth rates and reproduction in the spring fl owering species, Trillium erec- the understory herb Panax quinquefolius (Araliaceae). American Journal of tum (Liliaceae). American Journal of Botany 89 : 500 – 505 . Botany 94 : 819 – 826 . Rustad , L. E. , J. L. Campbell , G. M. Marion , R. J. Norby , M. J. Mitchell , A. E. Kaye , M. W. , and R. J. Wagner . 2014 . Eastern deciduous tree seedlings advance Hartley , J. H. C. Cornelissen , and J. Gurevitch . 2001 . A meta-analysis of the spring phenology in response to experimental warming, but not wetting, response of soil respiration, net nitrogen mineralization, and aboveground treatments. Plant Ecology 215 : 543 – 554 . plant growth to experimental ecosystems warming. Oecologia 126 : 543 – 562 . 1624 • AMERICAN JOURNAL OF BOTANY

Sage , R. F. , and D. S. Kubien . 2007 . Th e temperature response of C3 and C 4 Verheyen , K. , O. Honnay , G. Motzkin , M. Hermy , and D. R. Foster . 2003 . photosynthesis. Plant, Cell & Environment 30 : 1086 – 1106 . Response of forest plant species to land-use change: A life-history trait- Schimel , J. , T. C. Balser , and M. Wallenstein . 2007 . Microbial stress- based approach. Journal of Ecology 91 : 563 – 577 . response physiology and its implications for ecosystem function. Ecology 8 8 : Vitasse , Y. 2013 . Ontogenic changes rather than difference in temperature 1386 – 1394 . cause understorey trees to leaf out earlier. New Phytologist 198 : 149 – 155 . Schulz , K. E., and M. S. Adams . 1995 . Eff ect of canopy gap light environment on Vitasse , Y. , A. J. Porté , A. Kremer , R. Michalet , and S. Delzon . 2009 . Responses evaporative load and stomatal conductance in the temperate forest understory of canopy duration to temperature changes in four temperate tree species: herb Aster macrophyllus (Asteraceae). American Journal of Botany 82 : 630 – 637 . Relative contributions of spring and autumn leaf phenology. Global Change Schwartzberg , E. G. , M. A. Jamieson , K. F. Raff a , P. B. Reich , R. A. Montgomery , Biology 161 : 187 – 198 . and R. L. Lindroth . 2014 . Simulated climate warming alters phenological Walker , M. D. , C. H. Wahren , R. D. Hollister , G. H. R. Henry , L. E. Ahlquist , synchrony between an outbreak insect herbivore and host trees. Oecologia J. M. Alatalo , M. S. Bret-Harte , et al. 2006 . Plant community responses to 175 : 1041 – 1049 . experimental warming across the tundra biome. Proceedings of the National Seber , G. A. F. , and C. J. Wild . 1989 . Nonlinear regression. Wiley, New York, Academy of Sciences, USA 103 : 1342 – 1346 . New York, USA. Wang , H. , J. Dai , J. Zheng , and Q. Ge . 2015 . Temperature sensitivity of plant Sendall , K. M. , P. B. Reich , C. Zhao , H. Jihua , X. Wei , A. Stefanski , K. Rice , phenology in temperate and subtropical regions of China from 1850 to 2009 . R. L. Rich , and R. A. Montgomery . 2015 . Acclimation of photosynthetic International Journal of Climatology 35 : 913 – 922 . temperature optima of temperate and boreal tree species in response to ex- Wolkovich , E. M. , B. I. Cook , J. M. Allen , T. M. Crimmins , J. L. Betancourt , S. perimental warming. Global Change Biology 21 : 1342 – 1357 . E. Travers , S. Pau , et al. 2012 . Warming experiments underpredict plant

Taylor , R. J. , and R. W. Pearcy . 1976 . Seasonal patterns of CO2 exchange char- phenological responses to climate change. Nature 485 : 494 – 497 . acteristics of understory plants from a deciduous forest. Canadian Journal Woodward , F. I. , and B. G. Williams . 1987 . Climate and plant distribution at of Botany 54 : 1094 – 1103 . global and local scales. Vegetatio 69 : 189 – 197 .