Annals of Botany Page 1 of 13 doi:10.1093/aob/mcw162, available online at www.aob.oxfordjournals.org

Two tropical show strong growth and water-use efficiency responses to altered CO2 concentration

James W. Dalling1,2,*, Lucas A. Cernusak3, Klaus Winter2, Jorge Aranda2, Milton Garcia2, Aurelio Virgo2, Alexander W. Cheesman3, Andres Baresch4, Carlos Jaramillo2 and Benjamin L. Turner2 1Department of Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA, 2Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancon, Republic of , 3College of Science and Engineering, James Cook University, Cairns, Queensland 4870, Australia and 4School of Earth, Energy and Environmental Sciences, Stanford University, Stanford, CA 94305, USA *For correspondence. E-mail [email protected] Downloaded from

Received: 1 April 2016 Returned for revision: 17 May 2016 Accepted: 7 June 2016

Background and Aims Conifers dominated wet lowland tropical forests 100 million years ago (MYA). With a

few exceptions in the and Araucariaceae, conifers are now absent from this biome. This shift to http://aob.oxfordjournals.org/ angiosperm dominance also coincided with a large decline in atmospheric CO2 concentration (ca). We compared growth and physiological performance of two lowland tropical angiosperms and conifers at ca levels representing pre-industrial (280 ppm), ambient (400 ppm) and Eocene (800 ppm) conditions to explore how differences in ca af- fect the growth and water-use efficiency (WUE) of seedlings from these groups. Methods Two conifers (Araucaria heterophylla and guatemalensis) and two angiosperm trees (Tabebuia rosea and Chrysophyllum cainito) were grown in climate-controlled glasshouses in Panama. Growth, photosynthetic rates, nutrient uptake, and nutrient use and water-use efficiencies were measured. Key Results Podocarpus seedlings showed a stronger (66 %) increase in relative growth rate with increasing ca relative to Araucaria (19 %) and the angiosperms (no growth enhancement). The response of Podocarpus is consist- at University Of Illinois Library on August 31, 2016 ent with expectations for species with conservative growth traits and low mesophyll diffusion conductance. While previous work has shown limited stomatal response of conifers to ca, we found that the two conifers had signifi- cantly greater increases in leaf and whole-plant WUE than the angiosperms, reflecting increased photosynthetic rate and reduced stomatal conductance. Foliar nitrogen isotope ratios (d15N) and soil nitrate concentrations indicated a preference in Podocarpus for ammonium over nitrate, which may impact nitrogen uptake relative to nitrate assimi- lators under high ca. Significance Podocarps colonized tropical forests after angiosperms achieved dominance and are now restricted to infertile soils. Although limited to a single species, our data suggest that higher ca may have been favourable for podocarp colonization of tropical South America 60 MYA, while plasticity in photosynthetic capacity and WUE may help account for their continued persistence under large changes in ca since the Eocene.

Key words: Araucaria heterophylla, Podocarpus guatemalensis, Tabebuia rosea, Chrysophyllum cainito,tropical , angiosperm dominance, water-use efficiency, relative growth rate, nitrate assimilation, elevated CO2.

INTRODUCTION importance of these constraints diminishes at high latitude and elevation, where smaller conduit diameters reduce the risk of Tropical lowland forests are overwhelmingly dominated by freeze–thaw embolism, which can strongly impact stem con- angiosperm trees, representing a major transition from the con- ductivity (Feild and Brodribb, 2001; Lusk, 2011). iferous forests that began in the Cretaceous, and was largely Despite these apparent limitations on the competitive ability complete by the Paleogene 66 million years ago (MYA). The of lowland tropical conifers, two families, Araucariaceae and current dominance of angiosperms in lowland tropical forest is Podocarpaceae, remain locally important components of some generally considered to be at least in part a consequence of the forests, particularly on nutrient-poor soils (Dalling et al., 2011; inherently slow growth of conifer seedlings, which results in Enright and Jaffre´, 2011; Kitayama et al.,2011). Furthermore, competitive displacement by angiosperms unless light or soil recent work suggests that podocarps colonized tropical latitudes nutrient availability limits realized growth rates (Bond, 1989; only in the last 60 million years, and after the diversification of Midgley and Bond, 1991; Coomes and Bellingham, 2011; major angiosperm lineages (Jaramillo et al., 2011a, b; Morley, Brodribb et al.,2012). In turn, the slow growth of conifer seed- 2011; Quiroga et al.,2016). Ecological studies of extant trop- lings (but not necessarily larger individuals), can be attributed ical conifers therefore provide an opportunity to further explore to greater leaf mass per unit area and correspondingly lower the physiological characteristics of conifers that permit coexist- mass-based photosynthetic rates, vascular constraints on leaf ence with angiosperms under equitable tropical climates. In size, and narrow conduit diameters in conifer wood that limit addition, growth of these species under a range of atmospheric maximum conductance rates (Bond, 1989; Lusk, 2011). The CO2 concentrations (ca) allows us to explore how the relative

VC The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Page 2 of 13 Dalling et al. — Tropical conifer response to altered CO2 concentration growth advantage of angiosperms may have changed as ca Cerro Punta, Chiriqui, Panama, in early March 2010 and trans- declined at least 2-fold over the past 60 million years (Beerling ferred to Gamboa. Seedlings were grown for 3 months under and Royer, 2011; Franks et al.,2014), and under conditions of 30 % full sun prior to starting the experiment. Recently germi- contemporary climate change. nated seedlings of Podocarpus guatemalensis (Podocarpaceae) Here we examine the responses of seedlings of two tropical were collected from lowland forest at Playa Hermosa, Coiba 0 0 lowland conifers and angiosperms grown under ca representing Island, Panama (7 10 N, 81 32 W) and transplanted to pots in pre-industrial, ambient and Eocene levels (280, 400 and Gamboa in January 2010. They were grown under the same 800 mmol mol1 respectively) (Franks et al.,2014). We use shadehouse conditions for 5 months before the experiment these data to test two hypotheses. First, we examine whether began. Henceforth, species are referred to by genus name. tropical conifers show a stronger photosynthetic and growth re- Species were selected to match, as far as possible, seed mass sponse to variation in ca than angiosperms. This responsiveness and shade tolerance. Chrysophyllum and Podocarpus are both may be mediated by mesophyll conductance. Niinemets et al. relatively shade-tolerant, maintaining seedling banks beneath (2011) argued that mesophyll conductance, the capacity of CO2 closed canopy forest (J. W. Dalling, pers. obs.). Seedlings of to diffuse from sub-stomatal cavities to carboxylation sites in-

Tabebuia are relatively light-demanding (Kitajima, 2002), Downloaded from side chloroplasts, represents a larger fraction of the total limita- while Araucaria seedlings are likely to be at least moderately tion of diffusion to photosynthesis in sclerophyllous , shade-tolerant based on the ecology of congeneric species where thick cell walls strongly limit CO2 conductance. The (Enright, 1982; Duarte et al., 2002). Chrysophyllum, limitation imposed on photosynthesis by mesophyll conduct- Podocarpus and Tabebuia also co-occur in forests of northern ance can be partly overcome by increased ca irrespective of Coiba Island (Perez et al.,1996), and are widely distributed stomatal conductance. Although Niinemets et al. (2011) through . No Araucaria species are native to http://aob.oxfordjournals.org/ emphasized that mesophyll conductance limitation would par- Central America; A. heterophylla is native to Norfolk Island in ticularly affect evergreens that are adapted to drought, similar the tropical South Pacific. It differs from the other species in arguments apply to coniferous species that are adapted to low- having imbricate scale-like leaves, resulting in less efficient nutrient environments and that also build tough, long-lived light capture in forest understories (Coomes and Bellingham, leaves. 2011). Both Tabebuia and Chrysophyllum have been used ex- Second, we test whether tropical angiosperms are better able tensively in previous ecophysiological work in Panama (e.g. to optimize water-use efficiency than tropical conifers. Cernusak et al.,2008, 2011). All four genera form arbuscular Brodribb et al. (2009) argued that major clades of plants differ mycorrhizal associations (Dickie and Holdaway, 2010). at University Of Illinois Library on August 31, 2016 in water-use efficiency, representing a slow acquisition over 450 million years of the complex mechanisms that regulate guard cell apertures and balance mesophyll CO2 concentrations Seedling growth experiment and transpiration losses. In support of their argument, Brodribb In May 2010, 24 seedlings of each species were transplanted et al. (2009) cite meta-analyses that suggest different CO sen- 2 into 19-L pots containing a 1:1 by volume mix of soil and rice sitivity of gas exchange parameters of angiosperms and coni- husks (added to improve drainage). The soil was collected from fers, and show experimentally that the aperture of angiosperm an area formerly under lowland tropical forest and now in a pri- stomata, but not conifer stomata, are reduced significantly in re- vate orchard, allowing the removal of a large quantity of soil sponse to c of 600 mmol mol1 (but see Ward et al., 2013). a for experimentation. Soil was air-dried (27 C) and passed Differences in the stomatal responses reported by Brodribb through a 10-mm sieve. Details of the soil used in the experi- et al. (2009) appear to be a consequence of the evolution of ment and its analysis are described in Dalling et al. (2013). Ca2þ-dependent stomatal signalling (Brodribb and McAdam, Briefly, the soil has a high concentration of exchangeable phos- 2013). Nonetheless, stomatal conductance responses can be af- phate (17 8mgPkg1), based on extraction from anion ex- fected by plant age and by the duration of the study (Medlyn change membranes, and a high effective cation exchange et al. 2001), while instantaneous changes in ca miss the poten- 1 capacity (53 2 cmol kg ) based on extraction in 0 1 M BaCl tial for photosynthetic acclimation to CO , which may depend c 2. 2 Total KCl-extractable inorganic N availability is on the c under which leaves develop. In this experiment we a 62 5mgNkg1, but was reduced by the addition of the rice were able to assess growth, allocation and physiological husks (Dalling et al., 2013), counteracting the observation of responses after plants were grown under the same ca for 6–7 months. excess N mineralization observed in un-amended soils (e.g. Johnson et al.,1995; Ross and Hales, 2003). At the time of seedling transplant, three additional seedlings of each species MATERIALS AND METHODS were harvested to determine initial dry mass and leaf area. Eight pots per species were randomly assigned to one of Study species three glasshouses maintained at pre-industrial (280 ppm), ambi- In February 2010, seeds of the two angiosperm species, ent (400 ppm) and twice-ambient (800 ppm) atmospheric CO2 Tabebuia rosea (Bignoniaceae) and Chrysophyllum cainito concentration (Fig. 1). Transplanted seedlings were grown for (Sapotaceae), were collected from lowland forests in the 189–202 d under the treatment CO2 concentrations before har- Panama Canal area, and germinated in a shadehouse at the vest. The air in the treatment chambers was well mixed using Smithsonian Tropical Research Institute in Gamboa, Panama four 18-inch floor fans (McMaster-Carr, Robbinsville) in each 0 0 (9 07 N, 79 42 W). Seedlings of Araucaria heterophylla chamber. Control of CO2 concentrations was based on (Araucariaceae) were purchased from a commercial nursery in GMW21D carbon dioxide transmitters (Vaisala, Helsinki) and Dalling et al. — Tropical conifer response to altered CO2 concentration Page 3 of 13 a CR-5000 measurement and control system (Campbell whenever CO2 exceeded 280 ppm, air inside the chamber was Scientific, Logan, UT). For the 800-ppm ca treatment, injection sucked through an acrylic glass column (10 cm diameter, of pure CO2 was initiated when the CO2 concentration in the 100 cm height) filled with CO2 absorber up to a height of chamber fell below 795 ppm and was terminated when CO2 40 cm (Sofnolime 812 Mesh, Molecular Products, Boulder) concentration reached 800 ppm. The CO2 concentration was using the air pump and dustbag of an industrial vacuum cleaner prevented from overshooting by providing CO2 in multiple (Shop-Vac 125 Gal Peak HP, Novey, Panama). The air pump pulses of 2 s interrupted by 5 s without CO2 injection. To pre- was switched off when CO2 concentration reached 275 ppm. vent CO2 concentration from decreasing significantly below Light conditions were maintained at 50 % full sun using 400 ppm due to the photosynthetic activity of plants in the Aluminet Shade Cloth to cover the top of the chambers. The current-ambient CO2 treatment, during daytime, pulsed CO2 in- glasshouses were air-conditioned, with the air conditioners pro- jections were initiated when CO2 concentration fell below grammed to turn on when air temperature exceeded 30 C. 380 ppm and terminated when CO2 concentration reached Mean air temperature and humidity, recorded every 15 min dur- 400 ppm. For the below-current-ambient CO2 treatment, ing the experiment with a data logger (CR5000; Campbell Downloaded from

A http://aob.oxfordjournals.org/ at University Of Illinois Library on August 31, 2016

B C

FIG. 1. Glasshouse showing soda lime column and vacuum pump in foreground (A), seedling of Araucaria heterophylla (B) and (C) seedling of Podocarpus guatemalensis. Page 4 of 13 Dalling et al. — Tropical conifer response to altered CO2 concentration

Scientific, Logan, UT, USA), remained very similar across the Mass of foliage, stems and roots of harvested seedlings was three glasshouses (mean daytime temperature range 279– measured after drying for 72 h at 70 C. A sample of dried ma- 283 C; night-time temperature 253–267 C; daytime relative ture leaf material from each seedling was ground to a fine pow- humidity 609–613 %; night-time relative humidity 868– der for chemical and isotopic analyses using a Cyclotec 1093 893%). mill with a 05-mm screen (FOSS, Eden Prarie, MN, USA). Five additional pots without seedlings were included in Foliar N concentrations were determined by automated com- each glasshouse to determine soil evaporative water loss for cal- bustion and thermal conductivity detection on a Thermo Flash culations of whole-plant water-use efficiency (WUEwp, EA112 analyser (CE Elantech, NJ, USA) and foliar P by ashing 1 g dry mass kg water transpired). All experimental pots were at 550 C, followed by dissolution in 1 M H2SO4, with phos- watered to field capacity and allowed to drain overnight at the phate detection by automated molybdate colorimetry using a initiation of the experiment. The drain holes at the bases of the Lachat Quikchem 8500 (Hach Ltd, Loveland, CO, USA). In pots were then sealed for the duration of the experiment, and addition, at harvest time, soil was sampled from each pot con- the pots were weighed and re-watered to near their initial taining a seedling, and KCl-extractable nitrate and ammo- nium concentrations were measured after extraction in 2 M KCl weight each week, or at shorter intervals, as necessary. Pots Downloaded from were weighed to the nearest 5 g with a 64-kg-capacity balance by automated colorimetry using a Lachat Quikchem (Sartorius QS64B; Thomas, Swedesboro, NJ, USA). 8500 (Hach Ltd, Loveland, CO, USA), and available P was Cumulative plant water use (CWU) over the course of the ex- determined by extraction in Bray solution (Bray and Kurtz, 1945). periment was calculated as the sum of pot water loss minus the Foliar dry matter was analysed for its stable carbon isotope average sum of water loss from the control pots that did not 13 15 (d C) and stable nitrogen isotope (d N) ratios. Analyses were http://aob.oxfordjournals.org/ hold plants. Mean transpiration rate (MTR), calculated as 2 1 carried out in the Stable Isotope Laboratory of the Smithsonian kg m leaf area d , was calculated following Sheshshayee Tropical Research Institute with an elemental analyser (CE et al. (2005): Instruments, Milan, Italy) coupled to an isotope ratio mass spectrometer (Delta V; Thermo Fisher Scientific, Bremen, CWU 13 15 MTR ¼ (1) Germany). The d Candd N values are expressed relative to ðLA2 þ LA1Þ05t standards of PeeDee Belemnite and air, respectively. The car- bon isotope discrimination (D13C) of leaf dry matter was calcu- 13 13 where LA1 and LA2 are the initial and final leaf areas respect- lated as D C ¼ (da dp)/(1 þ dp), where da is the d CofCO2 13 at University Of Illinois Library on August 31, 2016 ively, and t is the duration of the experiment. in air in each glasshouse and dp is the d C of leaf carbon. The The WUEwp over the duration of the experiment was calcu- da was estimated by growing two species of C4 plant, lated for each plant as final dry mass minus initial dry mass Saccharum spontaneum and Portulaca oleracea, in each glass- 13 plus leaf litter produced divided by its CWU. house and outside in the open air. The d CofsourceCO2 for During the week preceding harvest, leaf gas exchange was plants grown outside was assumed to be –8‰, which was used 13 13 measured between 08:00 and noon on one leaf per plant with a to calculate the D CforeachC4 species. This D CoftheC4 portable photosynthesis system (Li-Cor 6400, Li-Cor Inc., species was assumed to be the same in the glasshouses and was Lincoln, NE, USA). These measurements were made at an ir- used to back-calculate da in each glasshouse. Thus, da was cal- radiance of 1200 mmol photons m2 s1, supplied by an artifi- culated as: cial light source attached to the leaf cuvette (6400-02B LED, 13 Li-Cor Inc.). The environment within the leaf cuvette was con- da ¼ D CC4ð1 þ dpC4ÞþdpC4 (2) trolled to be similar to ambient glasshouse conditions. Leaf temperatures during the measurements were between 28 and where subscript C4 refers to either S. spontaneum or P. olera- 35 C. Hereafter, we refer to these area-based net photosynthe- cea. By this method, we estimated a da of –82 ‰ in the sis rates as Amax and to the ratio of intercellular to ambient CO2 280 ppm CO2 glasshouse, –79 ‰ in the 400 ppm CO2 glass- concentration as ci/ca. These gas exchange data were also used house and –61 ‰ in the 800 ppm CO2 glasshouse. to calculate leaf-level WUE as net photosynthesis divided by Relative growth rate (RGR, mg g1 d1) was calculated as 1 transpiration (WUEleaf, mmol CO2 mmol H2O). For measure- the difference between ln(final dry mass) and ln(initial dry ments of Araucaria, with its scale-like leaves, a section of shoot mass) divided by the number of days in the experiment. Net as- was placed inside the 23 cm cuvette for the measurement, similation rate (NAR; biomass increment per unit leaf area, 2 1 and then immediately harvested for determination of the gm d ) was calculated for individual plants according to amount of leaf area that had been contained within the cuvette. the following equation: This was accomplished using an automated leaf area meter (LI- 3000A, Li-Cor Inc.). The same leaf area meter was used to ðWf WiÞðlnAf lnAiÞ NAR ¼ (3) measure the total leaf area of all seedlings following harvest. In tðAf AiÞ the case of Araucaria, however, whole-plant leaf area was underestimated as the minute and recurved leaves meant we where Wf and Wi are the final and initial dry mass (g), respect- 2 could only measure the projected area of individual branches. ively, Af and A i are the final and initial leaf area (m ) respect- Calculated values of specific leaf area and net assimilation rate ively, and t is the duration of the experiment (d). Leaf mass (see below) are nonetheless included for within-species com- fraction (LMF; leaf mass per unit whole plant mass, g g1), parison of ca treatment effects. stem mass fraction (SMF; stem and petiole mass per unit whole Dalling et al. — Tropical conifer response to altered CO2 concentration Page 5 of 13

90 15 A B 80

70 water) –1 60 10 mmol 50 2 40

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FIG. 2. Response of (A) final biomass and (B) water-use efficiency (WUE) of two angiosperm species (Chrysophyllum, open circles; Tabebuia, open triangles) and two conifer species (Araucaria, black squares; Podocarpus, black triangles) to growth at three levels of atmospheric CO2 concentration (280, 400, 800 ppm, log scale). Values are means 6 1 s.e. http://aob.oxfordjournals.org/ plant mass, units g g1), root mass fraction (RMF; root mass RESULTS 1 per unit whole plant mass, g g ), leaf area ratio (LAR; leaf Overall, we observed a stronger enhancement of both growth area per unit whole plant mass, cm2 g1) and specific leaf 2 1 (Fig. 2A)andWUE(Fig. 2B) in the conifers than in the angio- area (SLA; leaf area per unit leaf mass, cm g ) were calcu- sperm taxa in response to increasing c . lated from the final harvest data. Photosynthetic nitrogen use ef- a ficiency (PNUE; net carbon assimilation per unit leaf nitrogen, 1 1 mmol CO2 mmol N s ) was calculated from Amax measure- Growth and allocation at University Of Illinois Library on August 31, 2016 ments and foliar nitrogen concentration. The youngest fully expanded leaves were sampled in the Species showed contrasting growth responses to CO2 concen- weeks preceding harvest for measurements of stomatal density. tration (Figs 2A and 3A, Table 1). The angiosperms showed no Leaf area was measured (as above), and an impression of each net increase in RGR between 280 and 800 ppm, and did not dif- leaf was taken in the interveinal area of the lamina using clear fer statistically in response from Araucaria, which showed a fingernail polish. Impressions were taken only from the abaxial stimulation of 19 % in RGR. However, Podocarpus differed sig- surface of the leaves. The impressions were mounted on slides nificantly in growth response from the angiosperms (t ¼ 35, and photographed with a light microscope (Nikon Eclipse 200) P ¼ 0024), with 66 % stimulation in RGR from the lowest to at 200 to 400 magnification. Three fields were observed for the highest ca treatment (Fig. 3A). This represented a change in each leaf. Podocarpus stomatal density was estimated in two the mean plant biomass at harvest from 58 g in the lowest ca sections at the widest region of the blade; each measurement treatment to 234 g in the highest ca treatment (Fig. 2A). covered an area from the mid vein to the leaf margin (averaging Differences in growth performance were not related to differ- 2 87mm). In Araucaria, leaves have narrow bands of stomata ences in allocation. There were no significant species ca inter- separated by wide veins. Stomatal densities were calculated actions for leaf mass ratio (LMR), root mass ratio (RMR) or excluding non-stomatal areas. SLA (Table 1, Fig. 3B–D). Similarly, there were no significant differences among species in the response of NAR to ca (Fig. 3E), although the increase in mean NAR from the lowest to the Statistical analysis highest ca was greater for Araucaria (41 %) and Podocarpus (118 %) than for Chrysophyllum (28 %) and Tabebuia (26 %). Seedlings receiving the same ca were all grown in the same glasshouse and therefore shared the same growth environment. Increases in Amax perunitleafareainresponsetoca did not dif- Consequently, individual seedlings are not true experimental fer across species for Araucaria, Chrysophyllum and Podocarpus, but appeared to saturate at ambient ca in Tabebuia, replicates and only mean response values for each species ca were used in analyses. To evaluate species performance differ- the species with the highest SLA (Fig. 3F). In contrast, Podocarpus showed a very strong enhancement of Amax across ences across treatments, ca was treated as a continuous variable after log transformation. Response variables were also log- ca treatments, with a >4-fold increase from 280 to 800 ppm, transformed, as necessary, after checking for a linear relation- compared with a 2-fold increase in the remaining species. ship with ca.Twoaprioricontrasts were made of the species c interaction. First, we compared the two angiosperm and two a Nutrient relations conifer species. Second, the broad-leaved conifer, Podocarpus, was compared with the two angiosperms. Analyses were per- Foliar N and P (Table 1, Supplementary Data Fig. 1) differed formed in R version 2153(RCoreTeam,2013). significantly among species, but did not vary consistently Page 6 of 13 Dalling et al. — Tropical conifer response to altered CO2 concentration

40 0·8 AB

) 30 0·6 –1 d –1 20 0·4 Leaf mass ratio

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FIG. 3. Response of (A) relative growth rate (RGR), (B) leaf mass ratio, (C) root mass ratio, (D) specific leaf area, (E) net assimilation rate (NAR) and (F) maximal photosynthetic rate (Amax) of two angiosperm species (Chrysophyllum, open circles; Tabebuia, open triangles) and two conifer species (Araucaria, black squares; Podocarpus, black triangles) to growth at three levels of atmospheric CO2 concentration (280, 400, 800 ppm, log scale). Values are means 6 1s.e.

between the two species groups. Podocarpus and Tabebuia had significant species ca interaction for extractable nitrate the highest foliar N concentration, but conversely the lowest fo- (Table 1), with higher nitrate concentrations in pots containing liar P concentration. For all species, foliar N concentration the conifers (Fig. 4C). In addition, pots that contained 15 declined significantly with increasing ca, whereas foliar P either Podocarpus seedlings maintained a higher foliar d N across ca remained unchanged with ca (Podocarpus and Tabebuia)or treatments (Fig. 4E). Finally, the angiosperm taxa had higher increased significantly (Araucaria and Chrysophyllum). Foliar PNUE and showed greater increases in PNUE with ca (Fig. 4F) N:P suggested that all species became progressively more N- despite having foliar N:P values that were intermediate between and (Fig. 4A). limited at higher ca (Fig. 4A), although species did not show Araucaria Podocarpus differential responses of N:P to ca (Table 1). Consistent with N limitation, soil-extractable P remained high in all treatments and species (range 6–23 mg P kg1; Fig. 4B), while soil-ex- Water-use efficiency tractable ammonium and nitrate were reduced to low levels, Species differed significantly in MTR over the course of the particularly at 800 ppm ca (Fig. 4C, D). There was also a experiment, with a large decline for Araucaria with increasing Dalling et al. — Tropical conifer response to altered CO2 concentration Page 7 of 13

The slope of the relationship between log(ca – ci)and TABLE 1. F-values for species (d.f. ¼ 3), ca (d.f. ¼ 1) and species c (d.f. ¼ 3) effects on dependent variables for four tree species WUEwp was significantly greater for the two conifer species a than the two angiosperm species (d.f. ¼ 1,89, F ¼ 136, and three levels of ca. Where the species ca interaction is sig- nificant, contrasts between angiosperm and conifer taxa are re- P < 0001; Fig. 6), indicating that a mechanism additional to ca – ci contributed to the stronger responsiveness of WUEwp to ported in the text 13 ca in the conifers. Leaf dry matter D C and stomatal conduct-

Variable Species ca Species ca ance did not show a species ca interaction (Fig. 5D, E, Table 1), although both D13C(d.f. ¼ 1,8, F ¼ 841, P < 0001) and Growth and allocation conductance (d.f. 1,8, 16 5, 0 003) were significantly RGR 4079*** 223*** 114* ¼ F ¼ P ¼ NAR 798*** 354*** 096 lower for conifers than angiosperms. Finally, the stomatal dens- LMR 5319*** 159* 08 ity response showed a significant species ca interaction (Fig. RMR 1504*** 5005 5F, Table 1), attributable to a significant reduction in density SLA 910*** 5004 with increasing c in Podocarpus compared with the angio- Nutrient relations a sperms (t 6 3, P 0 008). Reduced stomatal density at high Foliar [N] 275** 158* 06 ¼ ¼ Downloaded from PNUE 1609*** 1341*** 57 ca in Podocarpus was also associated with a significant increase d15N66* 158* 06 in leaf size, from 59 613cm2 (mean 6 1 s.d.) at 280 ppm to Foliar [P] 686*** 302** 65* 99 617cm2 at 800 ppm. Foliar N:P 2794*** 928*** 29 Bray extractable soil P 254** 2410 KCl-extractable soil NO3-N 3080*** 798*** 622***

DISCUSSION http://aob.oxfordjournals.org/ KC-extractable soil NH4-N 646762 Photosynthesis and water-use efficiency Photosynthetic and growth responses to variation in c Mean transpiration rate 140* 577 082 a WUEleaf 116* 1551*** 359* Consistent with our first hypothesis that tropical conifers would WUEwp 2743*** 10097*** 994*** show a stronger photosynthetic and growth response to vari- Amax 161** 580** 03 Stomatal conductance 210** 100* 29 ation in ca than angiosperms, we saw a significantly stronger ci/ca 1759*** 701** 171** growth stimulation across treatments in Podocarpus (a 66 % in- C isotope discrimination 376** 0310 crease in RGR) and a weaker, but not statistically significant, Stomatal density 18666*** 120* 168* enhancement in Araucaria (a 19 % increase). There was no positive response to increasing c in either of the two angio- at University Of Illinois Library on August 31, 2016 *P<005; **P<001; ***P<0001. a sperm species. The lack of a growth response of Tabebuia and Chrysophyllum to ca is consistent with previous experiments in ca and for Tabebuia between 400 and 800 ppm CO2 (Fig. 5A, which seedlings of tropical tree species were grown in unfertil- Table 1). The response of WUEwp to ca differed significantly ized soil (Lovelock et al.,1998; Winter et al., 2000; Cernusak between the species groups (Fig. 5B, Table 1), with a stronger et al.,2011). positive response of WUEwp to ca in the conifers than in the The causes of differential responses of plants to elevated ca angiosperms (t ¼ 836, P < 0001). A similar pattern was seen in greenhouse experiments have recently been revisited by Ali in the instantaneous measurements of WUEleaf taken in the sev- et al. (2013). They contrasted evidence from short-term pot ex- eral days before harvest (Fig. 2B, Table 1). There was a strong periments, where species with high RGR are found to be most correlation between WUEwp and WUEleaf across the dataset responsive to elevated ca (Poorter and Navas, 2003; Ko¨rner, (r ¼ 075, P < 0001, n ¼ 93), indicating consistency between 2006), with model predictions that species with low C alloca- the time-integrated measure of water-use efficiency, based on tion to leaf production (LMR), and low stomatal conductance cumulative water use and biomass production, and the instant- (gs), instantaneous PNUE and SLA will be most responsive. aneous measurement, based on CO2 and water vapour fluxes Differences between longer-term, model-predicted responses to measured on single leaves. ca and those observed in greenhouses were suggested to result The steeper increases in WUEleaf with increasing ca in the from a short-term advantage to fast-growing species able to rap- conifer species compared with the angiosperm species could be idly increase in leaf area during the quasi-exponential phase of explained by decreases in the ratio of intercellular to ambient seedling growth (Ali et al.,2013). CO2 concentrations (ci/ca) with increasing ca (Fig. 5C, Table 1). Although in our experiment plants grew during this rapid ini- In Araucaria, the decline in ci/ca was associated with both tial seedling growth phase, we nonetheless observed stronger decreasing stomatal conductance and increasing photosynthesis, growth enhancement in the two conifers with less than half the whereas in Podocarpus the decline in ci/ca mainly resulted RGR of the angiosperm taxa. In agreement with the predictions from increasing photosynthesis from 280 to 800 ppm ca. In con- of Ali et al. (2013), the conifers had markedly lower gs,PNUE trast, ci/ca remained similar or showed a smaller decline with and SLA, but had the highest LMR. Low LMR in Tabebuia,al- increasing ca in the two angiosperm species. The leaf dry matter though this species had the highest SLA, may therefore in part 13 D C results, which provide a time-integrated measure of ci/ca, explain its low responsiveness species to ca. generally supported this pattern (Fig. 5D), although D13Cdid An additional mechanism to account for the high responsive- not decrease as much as expected based on the change in in- ness of the conifers has been proposed by Niinemets et al. stantaneous ci/ca in the 800 ppm ca glasshouse. However, across (2011). While limited stomatal conductance is widely recog- 13 the full dataset, instantaneous ci/ca and leaf dry matter D C nized to limit photosynthetic rate, Niinemets et al. (2011) re- were reasonably well correlated (r ¼ 079, P < 0001, n ¼ 96). viewed the effects of mesophyll conductance in plants and Page 8 of 13 Dalling et al. — Tropical conifer response to altered CO2 concentration

16 30 AB 14 25 12 )

–1 20 10

8 15

Foliar N:P Foliar 6 10

4 Soil P (mg kg 5 2

0 0 6 40 Downloaded from CD ) 5 –1 )

–1 30 4

20 3 http://aob.oxfordjournals.org/

2 10 Soil nitrate (mg N kg Soil nitrate 1 Soil ammonium (mg N kg

0 0 3 300

E F at University Of Illinois Library on August 31, 2016 ) 250 2 –1 s –1 200

1 mol N 2

15 150 0 Leaf δ 100

–1 50 PNUE ( μ mol CO

–2 0 200 400 800 200 400 800

CO2 concentration (ppm) CO2 concentration (ppm)

15 FIG. 4. (A) Seedling foliar N:P ratio, (B) soil-extractable P, (C) soil-extractable nitrate, (D) soil-extractable ammonium, (E) foliar d N and (F) photosynthetic nitro- gen use efficiency of two angiosperm species (Chrysophyllum, open circles; Tabebuia, open triangles) and two conifer species (Araucaria, black squares; Podocarpus, black triangles) in response to growth at three levels of atmospheric CO2 concentration (280, 400 and 800 ppm, log scale). Values are means 6 1s.e. highlighted the limited internal diffusion conductance of scler- effect for allocational traits (LMR and SLA), suggesting a pri- ophyllous plants, including conifers, as providing a constraint marily physiological response to ca. Contrary to expectation, to photosynthetic rate that can be as large as that due to stoma- however, photosynthetic responses to increasing ca did not dif- tal conductance. Reduced mesophyll conductance in scler- fer across species, although the magnitude of increase in Amax ophyllous plants is in turn attributable to greater investment in in Podocarpus was twice that in Chrysophyllum and Tabebuia support tissue, particularly thicker cell walls, associated with (Table 1, Fig. 3). Likewise, there was no significant interaction greater leaf longevity (Niinemets et al., 2005). Low mesophyll for net assimilation rate, although NAR enhancement was conductance has been observed in the New Zealand podocarp much larger in Araucaria and Podocarpus (Fig. 3). Dacrydium cupressinum, to the extent that the diffusive con- straint on photosynthesis imposed by mesophyll exceeded that Differences in nutrient uptake and use efficiency imposedbystomata(DeLucia et al.,2003). Despite the strong growth response of Podocarpus to Pot environments often result in concentrations of inorganic increasing ca, there was no significant species ca interaction N that are orders of magnitude higher than those in field soil Dalling et al. — Tropical conifer response to altered CO2 concentration Page 9 of 13

1·6 12 AB 1·4 10

1·2 water) ) –1

–1 8

d 1 –2 0·8 6

0·6 4 (g dry mass kg MTR (kg m 0·4 wp 2 0·2 WUE 0 0

0·8 30 Downloaded from C D 0·7 25 0·6 20 0·5 a http://aob.oxfordjournals.org/ c

/ 0·4 15 i c 0·3 10 0·2 5 0·1 Carbon isotope discrimination

0 0

0·25 250

E F at University Of Illinois Library on August 31, 2016 ) –1 0·2 ) 200 s –2 –2

0·15 150

0·1 100

0·05 Stomal density (mm 50 Conductance (mol m

0 0 200 400 800 200 400 800

CO2 concentration (ppm) CO2 concentration (ppm)

FIG. 5. (A) Mean transpiration rate (MTR), (B) whole-plant water-use efficiency (WUEwp), (C) ratio of intercellular to ambient CO2 concentration (ci/ca), (D) carbon 13 isotope discrimination (D C), (E) stomatal conductance (gs) and (F) stomatal density in response to growth at three levels of atmospheric CO2 concentration (280, 400 and 800 ppm, log scale). Values are means 6 1 s.e. Symbols are as in Fig. 2.

due to in situ mineralization (Johnson et al.,1995; Dalling levels of ca, potentially suggesting a greater preference of or- et al.,2013). Here we added rice husks as a soil conditioner to ganic N or ammonium over nitrate as an N source (Ho¨gberg, stimulate microbial uptake of N. In a previous pot experiment, 1997; Mayor et al., 2014) when compared with Araucaria and rice husk addition reduced soil-extractable nitrate availability the angiosperm taxa. Consistent with the d15N data, soil nitrate 10-fold over 7 weeks (Dalling et al.,2013), providing soil con- concentrations were much higher in pots in which Podocarpus ditions more in keeping with the low (<5 mgNg1)instantan- seedlings were grown. Differences in nitrate pool size across eously extractable inorganic N measured in tropical lowland species may in part be attributable to differences in N demand forest soils (Turner and Romero, 2009). driven by plant size. At harvest, Podocarpus seedlings were Analysis of soil and foliar N concentrations at the end of the much smaller than those of the other species (mean dry mass experiment indicated that the conifers likely differ in N uptake 58 versus 313–529 g at 280 ppm; 234 versus 360–788gat patterns from the angiosperm taxa. Podocarpus had a signifi- 800 ppm), and may have resulted in lower N limitation in cantly higher foliar d15N than the remaining species across all Podocarpus relative to the other taxa. Nitrate concentrations, Page 10 of 13 Dalling et al. — Tropical conifer response to altered CO2 concentration

16 the highest and lowest values in the conifers (Fig. 4A). Despite Araucaria large differences in foliar N and P concentrations among spe- 14 Chrysophyllum cies, PNUE was 3- to 4-fold lower in the conifer taxa. Podocarpus 12 Tabebuia Furthermore, the angiosperms showed a stronger increase in water) PNUE with ca. Conifer leaves, however, are likely to be longer- –1 10 lived than the angiosperm leaves, and PNUE differences may be smaller, or reversed when integrated over leaf lifespans. 8

6 Optimization of water-use efficiency in conifers and angiosperms 4 Brodribb et al. (2009) argued that major clades of plants dif-

WUE (g dry mass kg 2 fer in their ability to optimize WUE, with only angiosperms capable of responding to above-ambient ca through reduced sto- 0 matal aperture. Evidence for these differences in CO sensitiv- Downloaded from 50 100 1000 2 ity are suggested from meta-analyses that show considerable ca–ci (ppm) variation in responses of stomatal conductance to elevated ca among species (Curtis and Wang, 1998), and a stronger reduc- FIG. 6. Relationship between the draw-down in CO2 concentration from ambient air to the intercellular air spaces (caci) and water-use efficiency for two conifer tion in conductance in temperate angiosperm than coniferous species (Araucaria and Podocarpus) and two angiosperm species trees (Medlyn et al., 2001). Furthermore, when Brodribb et al. (Chrysophyllum and Tabebuia). http://aob.oxfordjournals.org/ (2009) exposed leaves of plants grown at ambient ca to elevated (600 ppm) and low (100 ppm) CO2, they found that stomata of four coniferous species were significantly less responsive to c however, were also somewhat elevated in the pots containing a than those of five angiosperms. These differences in the stoma- Araucaria, even though Araucaria dry mass at harvest was tal closure response translated into a significantly larger in- similar to or larger than that of Chrysophyllum and Tabebuia crease in WUE at elevated c in angiosperms. (Fig. 2A). Temperate conifers have frequently been reported a Short-term exposure to altered c , however, may not reflect to show a strong preference for ammonium over nitrate uptake a the longer-term capacity of plants to regulate stomatal conduct- (Lavoie et al., 1992; Kronzucker et al., 1997; but see ance and WUE (Medlyn et al., 2001). Although our study was at University Of Illinois Library on August 31, 2016 Rothstein and Cregg, 2005). More recently, Araucaria has relatively short in duration (6 months), there were strong re- been reported to grow faster when provided with ammonium sponses of WUE in the conifer species, with much larger in- rather than nitrate (Garbin et al., 2006), while Phyllocladus creases in WUE observed at high ca than in the two angiosperm (Podocarpaceae) has been shown to preferentially take up am- taxa. Moreover, the responses were confirmed with both in- monium over nitrate in excised roots (Warren and Adams, 15 stantaneous gas exchange measurements (WUEleaf) and experi- 2007). Although we lack d N data for field-grown trees, the ment-long measurements of biomass production relative to congener of our study species, Podocarpus oleifolius, does cumulative water use (WUE ). Interestingly, a lack of stoma- have high foliar d15N compared with co-occurring tree species wp tal closure in response to growth at elevated ca was indeed in montane forest in Panama, consistent with the results found observed in Podocarpus in our experiment (Fig. 5E), in agree- here (K. Heineman, University of Illinois at Urbana- ment with the results of Brodribb et al. (2009).However,a Champaign, USA, unpubl. res.). higher WUE was achieved in this species through larger pro- It has recently been shown that assimilation of nitrate into or- portional increases in photosynthesis with increasing ca than in ganic N compounds in leaves of C3 plants can be inhibited by the other taxa (Fig. 3F). These data highlight the combined role elevated ca, leading to limitations on growth responses to ca of stomatal conductance and photosynthetic capacity in defin- associated with declining N status as ca increases (Bloom et al., ing WUE, and demonstrate a pitfall associated with predicting 2010, 2012). This inhibition of nitrate assimilation in C leaves 3 WUE responses to ca based only on short-term stomatal respon- by elevated ca appears to be linked to inhibition of photorespir- siveness. Stomatal densities showed contrasting patterns be- ation (Rachmilevitch et al., 2004). Plants that preferentially tween Podocarpus and the remaining taxa. Consistent with take up ammonium are unlikely to experience the same inhib- higher WUE, stomatal density was reduced at high ca in ition, because ammonium is mainly assimilated into organic Podocarpus but not in the angiosperms. Reduced stomatal compounds in roots (Peoples and Gifford, 1997), which are pre- density may reflect greater leaf size at high ca in this species sumably unaffected by any change in photorespiration in leaves (Carins Murphy et al.,2014). associated with elevated ca.Thus,ifPodocarpus, and to a lesser It is intriguing that both Podocarpus and Araucaria achieved extent Araucaria,reliedmoreonammoniumuptakethandid very similar increases in WUEleaf in response to ca,butbydif- the angiosperm taxa, this could provide an additional explan- ferent means. Podocarpus mainly increased photosynthesis, ation for the stronger growth responses in these two conifers. whereas Araucaria both increased photosynthesis and reduced Despite potential differences in uptake, neither foliar N nor P stomatal conductance. The overall consequence for ci/ca allocation differed significantly between the two species (Fig. 5C) and WUEleaf (Fig. 2B) was much the same in either groups. Foliar N:P declined significantly in all species with ca, case. indicating greater N limitation as plant size increased. The plot of WUEwp against caci (Fig. 6) shows that there Nonetheless, N:P varied substantially among species, with both may be additional mechanisms to photosynthesis and stomatal Dalling et al. — Tropical conifer response to altered CO2 concentration Page 11 of 13 conductance that allowed the conifers to achieve larger in- partly underlie angiosperm dominance at low latitude (Bond, creases in WUEwp with increasing ca than did the angiosperms. 1989). Replication of similar studies in unfertilized soils across The WUEwp can be decomposed into components as follows a broader group of tropical tree taxa will help resolve whether (Farquhar and Richards, 1984): ca responses of low-latitude conifers are consistent, and whether they can therefore help explain how podocarps colonized ð1 u Þðc c Þ 2 angiosperm-dominated tropical northern South America WUE ¼ c a i (4) wp 1 6v 3k 60 MYA (Quiroga et al.,2016). Results from this study are consistent with the expected where /c is the proportion of carbon gained through net photo- growth enhancement for evergreen species with low mesophyll synthesis that is subsequently lost from the plant by respiration, conductance (Niinemets et al.,2011), and more generally for 16 is the ratio of diffusivities of water vapour and CO2 in air, v slow-growing species with low carbon-use efficiency (Ali et al. is the leaf-to-air vapour pressure difference, 2/3 is the ratio of 2013). Alternatively, the growth response of the conifers may molecular weights of carbon and water, and k is the whole- reflect an advantage in nutrient (N) acquisition. Evergreen spe-

plant carbon mass fraction. The results shown in Fig. 6 in con- cies have increased in abundance in a range of ecosystems, al- Downloaded from junction with eqn (4) suggest that the combined term (1 /c)/ though the drivers of these changes are not always clear vk differed across the range of ca between the conifers and (Niinemets et al., 2011, and references therein). Podocarps are angiosperms, because the slope of the relationship between currently patchily distributed across the lowland and montane WUEwp and caci differed. A larger (1 /c) in the conifers tropics, and for the most part are restricted to low-fertility habi- could indicate increasing carbon-use efficiency as ca increased. tats, where soil nutrient availability constrains realized growth Further research is needed to tease these effects apart, but it is rate (Adie and Lawes, 2011; Dalling et al., 2011; Enright and http://aob.oxfordjournals.org/ apparent that multiple physiological mechanisms contributed to Jaffre´, 2011; Punyasena et al.,2011). Contemporary increases the stronger responsiveness of WUEwp to ca in the conifer com- in ca that improve the growth performance of tropical forest pared with angiosperm taxa. conifers relative to angiosperm trees might therefore be ex- An interesting result in our experiment was that the apparent pected to influence their abundance and distribution. D13 change in C from low to high CO2 was less in the conifer Although higher ca may have provided greater opportunities taxa than expected from the instantaneous measurements of ci/ for podocarps to expand their distributions when they first ca [compare panels (C) and (D) in Fig. 5]. We did not have dir- 13 colonized South America and tropical South-East Asia in the ect measurements of the d CofCO2 in air in the glasshouses, Palaeocene and late Eocene (Jaramillo et al., 2011a, b; Morley, at University Of Illinois Library on August 31, 2016 and so relied on the C method to assign these values (Marino 4 2011), high ca was not apparently a requisite for range expan- and McElroy, 1991). If there were a bias in this method at high 13 sion. Podocarps and the araucarian Cyclusphaera continued to CO2, it might have caused us to overestimate the d CofCO2 expand in range during the Oligocene (Jaramillo et al., 2013), in air in the 800 ppm ca glasshouse by 1–2‰. This could at when c was closer to current levels (Franks et al., 2014). least partly reconcile the variation in trends in panels (C) and a 13 Furthermore, podocarps show evidence of distributional shifts, (D) in Fig. 5. On the other hand, if our estimates of D Cwere potentially including the colonization of lowland forests, during accurate, when combined with the instantaneous measurements glacial maxima (e.g. Bush et al.,1990; Colinvaux et al.,1996) of ci/ca they would suggest that changes in stomatal conduct- when ca was <200 ppm (Monnin et al.,2001). Evidence pre- ance and mesophyll conductance were not proportional in re- sented here shows that extant tropical conifers exhibit consider- sponse to increasing CO2 in the conifer taxa. It is not known able plasticity in photosynthetic capacity and WUE to whether the response of mesophyll conductance to varying c a accommodate these changes in c . differs between conifers and angiosperms, but it has recently a been speculated that this could be an important consideration for predicting responses of these two groups of plants to rising SUPPLEMENTARY DATA c (Flexas et al.,2014). a Supplementary data are available online at www.aob.oxfordjour nals.org and consist of Figure S1: (A) foliar nitrogen concentra- Implications for understanding forest composition tion and (B) foliar phosphorus concentration of seedlings of the conifers Araucaria (black squares) and Podocarpus (black tri- In this study, the conifer Podocarpus guatemalensis,inpar- angles) and the angiosperms Chrysophyllum (open circles) and ticular, showed a strong growth response to ca, accumulating Tabebuia (open triangles) grown at 280, 400 and 800 ppm CO . 1 2 twice as much biomass at 400 mmol mol CO2 and more than 1 four times as much at 800 mmol mol CO2 compared with 1 ACKNOWLEDGEMENTS pre-industrial ca (280 mmol mol CO2). This result suggests that podocarps, and perhaps other low-latitude conifers, are We thank the National Environmental Authority of Panama likely to have experienced a significant enhancement of com- (ANAM) for permits and the Smithsonian Tropical Research petitive ability as ca has risen over the past century (and over Institute for logistic support at Coiba and Gamboa. the lifespan of individual trees). Although the physiological re- sponses of seedlings described here may not scale to larger size classes, or to evolutionary timescales, differences in seedling LITERATURE CITED growth rates between angiosperms and conifers may play an Adie H, Lawes MJ. 2011. Podocarps in Africa: temperate zone relicts or rainfor- important role in recruitment success, and are thought to at least est survivors? In: Turner BL, Cernusak LA, eds. Ecology of the Page 12 of 13 Dalling et al. — Tropical conifer response to altered CO2 concentration

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