Oecologia DOI 10.1007/s00442-014-2887-9
Physiological ecology - Original research
Gap effects on leaf traits of tropical rainforest trees differing in juvenile light requirement Nico C. Houter · Thijs L. Pons
Received: 6 June 2013 / Accepted: 11 January 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract The relationships of 16 leaf traits and their plasticity with the dependence of tree species on gaps for regeneration (gap association index; GAI) were examined in a Neotropical rainforest. Young saplings of 24 species with varying GAI were grown under a closed canopy, in a medium-sized and in a large gap, thus capturing the full range of plasticity with respect to canopy openness. Structural, biomechanical, chemical and photosynthetic traits were measured. At the chloroplast level, the chlorophyll a/b ratio and plasticity in this variable were not related to the GAI. However, plasticity in total carotenoids per unit chlorophyll was larger in shade-tolerant species. At the leaf level, leaf mass per unit area (LMA) decreased with the GAI under the closed canopy and in the medium gap, but did not significantly decrease with the GAI in the large gap. This was a reflection of the larger plasticity in LMA and leaf thickness of gap-dependent species. The well-known opposite trends in LMA for adaptation and acclimation to high irradiance in evergreen tropical trees were thus not invariably found. Although leaf strength was dependent on LMA and thickness, plasticity in this trait was not related to the GAI. Photosynthetic capacity expressed on each basis increased with the GAI, but the large plasticity in these traits was not clearly related to the GAI. Although gap-dependent species tended
Communicated by Fernando Valladares. Electronic supplementary material The online version of this article (doi:10.1007/s00442-014-2887-9) contains supplementary material, which is available to authorized users. N. C. Houter · T. L. Pons (*) Department Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Padualaan 8, 3508 CH Utrecht, The Netherlands e-mail: [email protected]
to have a greater plasticity overall, as evident from a principle component analysis, leaf traits of gap-dependent species are thus not invariably more phenotypically plastic. Keywords Acclimation · Gap dependence · Leaf trait · Plasticity · Shade tolerance
Introduction Light availability is low under the canopy of undisturbed evergreen tropical rainforest. Creation of a gap, such as that caused by fallen trees, increases plant exposure to the sky. The resulting increased irradiance has a direct effect on plant growth and it also drives changes in other microclimatic conditions such as elevated daytime temperatures and associated vapour pressure deficit (Chazdon et al. 1996). Gap conditions can be conducive for plant growth as a result of the increased light availability but can also be stressful to plants. Species may need canopy gaps or large disturbances for establishment and further development; these are referred to as ‘gap-dependent species’. However, high irradiance, especially in combination with high temperatures, can be stressful for shade-tolerant species that are capable of establishing under a closed canopy (Mulkey and Pearcy 1992; Lovelock et al. 1994; Houter and Pons 2005). Gap-dependent species and shade-tolerant species are not clearly distinct functional groups. The requirement of gaps for regeneration is better considered as a continuum (Popma and Bongers 1988; Osunkoya et al. 1994; Poorter et al. 2004; Coste et al. 2005; Laurans et al. 2012). Several studies have shown that functional leaf traits vary systematically with a species’ light requirement for regeneration (Valladares and Niinemets 2008). Species from evergreen tropical rainforest that are strongly
13
Oecologia
dependent on gaps for regeneration generally have a low leaf mass per unit area (LMA). Their leaves have short life spans and they are not well defended against herbivores. They are further characterized by low strength and high photosynthetic capacity (Bongers and Popma 1990; Reich et al. 1992; Raaimakers et al. 1995; Coley and Barone 1996; Westoby et al. 2002; Poorter and Bongers 2006). The leaves of shade-tolerant species have opposite characteristics. This is evidence for a trade-off between investment in defence, that is favoured in shade-tolerants, and in photosynthetic functions, as favoured in gap-dependent species (Mooney and Gulmon 1982; Coley et al. 1985). Plants can acclimate to the variation in the light climate as found under a closed canopy and in gaps. Numerous studies indicate that at the leaf level, plants growing at high irradiance compared to their counterparts in the shade have thicker leaves with a higher LMA and an associated higher photosynthetic capacity per unit leaf area (Lambers et al. 2008; Poorter et al. 2009). These leaves are also stronger (Onoda et al. 2008). These increases with acclimation to high irradiance are opposite to the decrease in LMA and leaf strength with adaptation to gap conditions in evergreen tropical trees as mentioned above (Veneklaas and Poorter 1998; Lusk et al. 2008). The picture is different for photosynthetic capacity per unit leaf area, because both acclimation and adaptation to high irradiance lead to higher values (Kitajima 1994; Raai-makers). Several studies indicate that leaf-level plasticity associated with acclimation to high irradiance is larger for gap-dependent species compared to shade-tolerant ones (Strauss-Debenedetti and Bazzaz 1996; Ellsworth and Reich 1996; Valladares et al. 2000); but this is not invariably found for all traits (Popma et al. 1992; Rozendaal et al. 2006). Traits at the chloroplast level, such as photosynthetic capacity per unit chlorophyll and pigment composition, can also be modified with acclimation to the level of irradiance. Photosynthetic capacity per unit chlorophyll is higher in high-light compared to shade-grown leaves. This is the result of a change in the organization of the chloroplasts (Anderson et al. 1995; Hikosaka and Terashima 1995). Photon absorption is not a limiting factor for photosynthesis at high irradiance, which makes a relatively large investment in photosynthetic capacity efficient in these light conditions. However, in shade environments, photon absorption limits photosynthetic rates and shade-acclimated leaves typically have more chlorophyll relative to capacity (Evans 1989; Hikosaka and Terashima 1996; Pons and Anten 2004). The chlorophyll a/b ratio pertains to the relative investment in the light harvesting complex of photosystem II (LHCII), which is rich in chlorophyll b. A low chlorophyll a/b, as found in many shade-grown leaves, thus represents a large LHCII, which is consistent with the low photosynthetic capacity per unit chlorophyll. However, not all tropical plants exhibit a clear increase of chlorophyll a/b with increasing light availability
13
(Krause et al. 2001; Matsubara et al. 2009). Several carotenoids are associated with photoprotection in high-light conditions and their contents per unit chlorophyll tend to increase with increasing irradiance; this also occurs in tropical trees (Krause et al. 2004; Krause et al. 2012). Relationships of these chloroplast-level trait values and their plasticity with species’ juvenile light requirement has not been systematically investigated in tropical trees so far. The first question was to what extent leaf traits are associated with the regeneration niche of tropical rainforest trees and what the relationships between the traits are, with particular respect to the chloroplast-level traits that are not well studied in evergreen tropical trees? Tree species were selected that covered a wide range of requirements for canopy gaps for their regeneration. Trait values were related to an index of gap dependence of a species, which is equivalent to the inverse of shade tolerance. Young saplings of the trees were grown in common gardens in the forest under a closed canopy and in gaps of two different sizes. The shade-tolerant and gap-dependent species were thus exposed to an identical full range of irradiance from deep shade to almost full daylight as opposed to sampling from naturally occurring populations where the range is more limited and not necessarily identical. This enabled us to investigate the species under conditions of canopy openness where they are not easily found. Growth of the plants in the full range of irradiance allowed the comparison of full plasticity in the different traits and to address the second question: how is the plasticity of the traits associated with a species’ regeneration niche?
Materials and methods Study site The study was carried out in a largely undisturbed forest in Central Guyana at about 50 km south of the Mabura Hill township in the West-Pibiri compartment of the local timber concession (5°02′N, 58°37′W). The dominant soil is a Haplic Ferralsol, locally known as ‘brown sandy soil’. It is a highly weathered acidic soil that has a high iron, aluminium and manganese content, and is low in available nutrients (van Kekem et al. 1996). Average annual precipitation is around 2,700 mm (Pons and Helle 2011). Approximately circular gaps of different sizes had been created in the forest in 1996 (van Dam 2001; Houter and Pons 2005). A medium-sized and a large gap were used for this study. Plant material Young saplings of 24 species that are common in the area were used for the study. Ten of those are among the
Oecologia Table 1 Species used in the study, their family, and their gap association index (GAI) value
a
Ranges from 1 to 5, where low values indicate that regeneration from seed is predominantly found under a closed canopy and in small gaps and high numbers indicate predominantly regeneration in large gaps and clearings. Each value is the mean (± SD) of five estimates
Species
Family (subfamily)
GAIa
Oxandra asbeckii (Pulle) R.E. Fr.
Annonaceae
1.0 ± 0.0
Tapura guianensis
Dichapetalaceae
1.5 ± 0.6
Mora gongrijpii (Kleinh.) Sandw.
Leguminosae (Caesalpinioideae)
1.6 ± 0.9
Eschweileria sagotiana Miers
Lecythidiaceae
1.7 ± 0.6
Chlorocardium rodiei (Schomb.) Rohwer. Richter and v.d. Werff
Lauraceae
1.8 ± 0.8
Lecythis concertiflora (A.C. Sm.) Mori
Lecythidaceae
1.8 ± 0.4
Vouacapoua macropetala Sandw.
Leguminosae (Caesalpinioideae)
1.8 ± 1.3
Protium guianensis var. guianensis
Burseraceae
2.1 ± 1.3
Catostemma fragrans Benth.
Bombacaceae
2.3 ± 0.7
Licania heteromorpha Benth. var. perplexans Sandw.
Chrysobalanaceae
2.3 ± 1.3
Ormosia coccinea (Aubl.) Jacks.
Leguminosae (Papilionoideae)
2.5 ± 0.7
Dicymbe altsonii Sandw.
Leguminosae (Caesalpinioideae)
2.6 ± 0.8
Parinaria campestris
Chrysobalanaceae
2.7 ± 0.6
Pouteria speciosa
Sapotaceae
2.7 ± 1.2
Hymenea courbaril L. var. courbaril
Leguminosae (Caesalpinioideae)
3.1 ± 1.3
Pentaclethra macroloba (Willd.) Kuntze
Mimosoideae
3.2 ± 0.4
Carapa guianensis Aubl.
Meliaceae
3.3 ± 1.4
Inga spp.
Leguminosae (Mimosoideae)
3.4 ± 1.1
Peltogyne venosa (Vahl) Benth. subsp. densiflora (Spruce ex Benth.) M.F. Silva
Leguminosae (Caesalpinioideae)
3.8 ± 1.0
Sclerolobium guianense Benth. var. guianense
Leguminosae (Caesalpinioideae)
4.6 ± 0.4
Goupia glabra Aubl.
Celastraceae
4.7 ± 0.4
Laetia procera (Poepp.) Eichler
Flacourtiaceae
5.0 ± 0.0
Jacaranda copaia (Aubl.) D. Don subsp. copaia
Bignoniaceae
5.0 ± 0.0
Cecropia obtusa Tréc.
Moraceae
5.0 ± 0.0
hyperdominant species distinguished by ter Steege (2013) across Amazonia, and another eight are members of genera that contain one or more hyperdominant. The selected species differ in their requirement of canopy gaps for their regeneration. To what extent they depend on gaps was characterized for each species in a similar way to Osunkoya et al. (1994) and Mostacedo and Fredericksen (1999). The species were assigned to one of five categories by one of us (N. C. Houter), an experienced tree spotter (S. Roberts), and three ecologists with long-standing working experience in the local forest (P. van der Hout, H. ter Steege and R. C. Zagt). The categories were based on the occurrence of seedlings and small saplings in canopy gaps (1) mostly under a closed canopy and occasionally in small gaps, (2) mostly in small gaps and under a closed canopy, (3) mostly in gaps of different sizes and occasionally under a closed canopy, (4) exclusively in gaps of different sizes and not under a closed canopy of undisturbed forest, (5) typically in large gaps and clearings. The results were averaged and the means were used as an index of association of a species as juveniles with canopy gaps [the gap association index (GAI)]. Species and
their GAI are shown in Table 1. This index is thus representative for a species’ light requirement for regeneration and is inversely related to its shade-tolerance. Experimental design and leaf sampling The effect of growth of young saplings in gaps in the forest canopy on leaf trait values was investigated by comparing them with counterparts growing under the closed forest canopy. The objective was to measure the full range of plasticity of a species with respect to canopy openness. As most species typically do not cover the full range of canopy openness in their natural habitat, they were planted in these two conditions. Shade-tolerant species may not perform well in a large gap and may not exhibit the maximum (or minimum) trait values there as in gaps of smaller size. Hence, in addition to the control conditions under the closed canopy, seedlings were planted in a large gap (ca. 2400 m2) and in a medium-sized gap (ca. 320 m2). Four species, Tapura, Vouacapoua, Dicymbe and Inga, were not planted in the medium gap. This approach allowed us
13
Oecologia
to compare species under identical conditions in the full range of canopy openness from deep shade under a closed canopy to almost full daylight in a large gap. Our experimental approach is positioned between traditional shade house experiments, where plants are typically grown in pots under spectrally neutral screens, and sampling from populations under the gradient of canopy openness, where they naturally occur. Contrary to shade house experiments, our plants experienced the natural variation in irradiance, spectral, directional and temporal components of the light environment, and variation in other environmental factors associated with variation in canopy openness such as temperature, wind and humidity. Also possible pot effects are excluded (Poorter et al. 2012). Seeds were locally collected and germinated in a nursery (4.7 % of full daylight). When seeds were not available, seedlings were collected in the surrounding forest. An area of 130 m2 in the gaps was prepared by excavating roots and removing larger debris, and clearing secondary growth before planting. Ten plants per species were randomly planted at a spacing of 0.8 m. It was necessary to nurse sensitive species after planting in the high-light environment of the gaps, as indicated by preliminary tests. All plants were shaded for 1 to 2 months until growth had resumed and watered in dry periods to avoid excessive water stress. Litter from the surrounding forest was put on top of the soil and the plants received initially 5 g of slow-release fertilizer (Osmocote plus; release time 2–3 months). Daily photosynthetic irradiance was around 35 and 90 % of above-canopy values in the medium and large gaps, respectively, as measured with quantum sensors connected to a data logger (LI192 s and LI-1000; LI-COR, USA). The seedlings that were planted under the closed canopy received about 1 % of daily above-canopy photosynthetic irradiance. Plots with planted seedlings were fenced against larger herbivores. The gapdependent species (GAI > 4; Table 1) that did not survive in sufficient numbers in the deep shade conditions under the closed canopy of the primary forest were sampled in the most shaded places under a closed canopy in disturbed areas where relative irradiance was between 2 and 3 %. Their full range of growth conditions was thus slightly different from that of the more shade-tolerant species. The plants were grown in the gaps for about 5 months when new leaves had formed under the gap conditions at the time of sampling. We attempted to keep plant height equal between species at sampling, but some variation between species could not be avoided (24 ± 14 cm; mean ± SD) as a result of variation in seed mass (more than 5 orders of magnitude) and growth rate. It could also not be avoided that some plants growing under the closed canopy were smaller than gap-grown plants, particularly the small-seeded gap-dependent species. Cotyledons were fully exhausted at the time of sampling, hence, plants were beyond the seedling stage.
13
Recently matured leaves were measured for thickness, area, fresh and dry mass, toughness, pigments, nitrogen and the light-saturated electron transport rate (ETR). Sampling was done between March and May 2000 when daily photosynthetic irradiance was around 28 mol m−2 day−1. Photosynthetic traits Photosynthetic capacity was measured as the light-saturated ETR by means of chlorophyll fluorescence. A pulse-amplitude-modulation fluorometer (mini-PAM, Walz, Effeltrich, Germany) was used with the leaf clip holder (2030-B) for measurement of the quantum yield of PSII in the light (ΦPSII = ΔF/Fm’; where Fm’ is maximum fluorescence in light) and photon flux density (PFD). Measurements in the gaps were done in the morning before or shortly after direct sunlight hit the sample leaf. Additional lighting with a halogen lamp was used to increase irradiance to saturating levels when required. The intensity of the measuring light and the irradiance required for light saturation in each species and light condition was established in preliminary measurements. Irradiance was reduced when ΔF/Fm’ was lower than 0.2, which was achieved by adjusting the irradiance, for better accuracy of the ETR measurement. The saturating pulse was set at its maximum value (6 mmol m−2 s−1). ETR was calculated according to Genty et al. (1989) as ETR = PFD × ΦPSII × α × β, where ΦPSII = ΔF/Fm’. The leaf absorbance (α) was derived from chlorophyll per unit area (chl; μmol m−2) as α = chl/(chl + 76) (Evans and Poorter 2001), and an equal partitioning of photon absorption between the two photosystems was assumed (β = 0.5). ETR thus calculated provides a good measure of gross photosynthesis when cyclic electron transport is of minor importance (Genty et al. 1989). Structural, chemical and biomechanical traits Whole leaves or leaflets that had been used for the photosynthesis measurements were stored between moistened filter paper until further processing the same day. In the case of large leaves, such as those of Cecropia, about 100 cm2 of the leaf blades was used. Leaf area was measured with a leaf area meter (LI-1100; Li-COR) and fresh mass. Dry mass was measured after 3 days at 70 °C. Leaf mass per area (LMA) was calculated from the dry mass to area ratio. Thickness of the fresh leaf was measured with a thickness gauge (resolution 0.01 mm) excluding the veins. Pigments were extracted from three leaf discs (1.9 cm2) in 5 ml dimethylformamide after storage in darkness at room temperature for 4–10 days when extraction was completed. Absorbance was measured with a spectrophotometer (Shimadzu UV/VIS 120-01; Shimadzu, Kyoto), and chlorophyll a and b, and total carotenoids were calculated
Oecologia
according to Inskeep and Bloom (1985). Total leaf nitrogen per unit dry mass was measured on ground leaf material using an elemental analyser (Carlo Erba, Milan) on closedcanopy and large-gap plants only. Leaf strength was measured on separate but similar leaves to those used for the previous measurements according to Feeny (1970) and Choong et al. (1992). A leaf was put between two acrylic plates; the lower one had a hole of 5 mm diameter and the upper one a hole of 2 mm diameter. A freely moving metal rod (cross-section 2.5 mm2) with a beaker on top rested perpendicularly on the leaf. The force on the leaf was slowly increased by filling the beaker with water. The total weight when the rod penetrated the leaf was used to calculate the punch strength by dividing it by the cross-sectional area of the rod. Calculations and statistical analyses The effect of growth in a gap was expressed as the relative change in a trait value per species when comparing gap-grown plants (gap) with plants grown under the closed canopy (CC): (gap–CC)/CC. Two-way ANOVAs were used to evaluate species specific differences in gap effects on leaf trait values after log-transformation. The relationships with the GAI of the species mean trait values per growth condition and the gap effects on the traits were evaluated by means of regression analysis. Correlations between leaf traits per growth condition and between gap effects on traits were evaluated with the Spearman correlation coefficient (twosided). The maintenance of species ranking between growth conditions was calculated by pair-wise comparison of the species mean trait values and its significance was evaluated by calculating Kendall’s correlation coefficient. Associations among leaf traits per growth condition and plasticity of traits were analysed with factor analysis extracted from a correlation matrix using principle component analysis (PCA). Sampling adequacy and sphericity were tested (KMO and Bartlett tests), and solutions were orthogonally rotated (varimax rotation) and limited to a maximum of two factors. Plasticity per trait was calculated from the maximum (max.) and minimum (min.) mean values according to Valladares et al. (2006) as: (max.–min.)/max. The gap effect on a trait value and the plasticity index were highly correlated (mean r = 0.97). The statistical analyses were carried out using SPSS 20 (IBM, Armonk, NY).
Results Plants grown under a closed canopy The PCA plot shows that the photosynthetic variables were strongly associated with the GAI on the first axis
(Fig. 1a). This illustrates the highly significant increase in photosynthetic capacity (ETR) expressed on each basis with increasing gap dependence of a species (Fig. 2; Table 2). However, the leaf traits pertaining to strength and structure, punch strength, LMA, density and thickness, occupied a position more to the left on the first axis (Fig. 1a), as evidence of their negative relationship with the GAI (Fig. 2; Table 2) and with the photosynthetic variables (Supplemental Table S4). Punch strengthLA (for abbreviations see Table 2) was positively correlated with LMA and thickness, but not with density (Supplemental Table S4). ChlorophyllLA was not related to a species’ gap dependence in closed-canopy growth conditions; nor were the other pigment variables, chlorophyll a/b ratio and carotenoidschl (Table 2; Fig. 2). Due to the decrease in LMA with the GAI, chlorophyllDM increased with the GAI (Table 2). The same is true for chlorophyllN (Fig. 2), which is the result of a stronger increase with the GAI of chlorophyllDM compared to NDM (Table 2). Leaves of shade-tolerant trees compared to those of gap-dependent species growing in dense canopy shade were thus characterised by a low photosynthetic capacity expressed on each basis. Their leaves were strong and had a high LMA, and tended to be thicker and denser. Their chlorophyllLA, chlorophyll a/b and carotenoidschl were not different from those of gap-dependent species, but their chlorophyllDM and chlorophyllN were lower. Their leaves had also a somewhat lower NDM and their NLA tended to be somewhat higher. The effect of growth in gaps Growth in gaps compared to growth under a closed canopy had highly significant effects on all measured leaf traits as evident from a two-way ANOVA (Supplemental Table S1). The highly significant species × gap size interactions indicate that species responded differently to the canopy gaps. Most trait values increased as a result of growth in a gap, but some decreased, such as chlorophyll, expressed on each basis, and punch strengthDM (Table 3). Most trait values showed the largest difference when comparing closed-canopy with large-gap-grown plants. However, the gap effect had already reached its maximum in the medium gap for some traits such as LMA and ETRLA for some shade-tolerant species, and for carotenoidschl for gap-dependent species (Figs. 2, 3; Supplemental Tables S2, S3). The effect of growth in gaps is mainly discussed by comparing large-gap plants with closed-canopy plants, as not all traits were measured on medium-gap plants and not all species were included (Table 1). Medium-gap plants are considered where appropriate.
13
Oecologia
Fig. 1 Principal component analysis (PCA) of leaf traits and their plasticity in 23 species (all species except Pentaclethra). a–c Loadings of the leaf traits on the first two components that were extracted from the correlation matrix. d–f Species factor scores on the first component plotted against their gap association index (GAI). The GAI was excluded from the analysis in the latter case. Juveniles grown a, d under a closed canopy, and b, e in the large gap; c, f plasticity index for the large-gap and closed-canopy trait value comparison [(mean maximum value–mean minimum value)/mean maximum
value]. a–c Variance explained by each of the components in parentheses (for axis 2 above the axis). The second percentage refers to the PCA without the GAI. Dry mass-based traits were excluded where leaf area-based traits were available. Also, chlorophyll a/b was not included as it was not related to the GAI and reduced the explained variance of the model. thick Thickness, dens density, strength punch strength, chl chlorophyll, carot carotenoidschl, A light-saturated electron transport rate, Tg Tapura, Co Cecropia
There was a similar clustering in the PCA plot of the large-gap compared to closed-canopy-grown plants (Fig. 1a, b) and regressions of trait values on the GAI showed also several similarities (Table 2). The similarity is also expressed in the maintenance of the species ranking in the traits for large gap compared to closed-canopy-grown plants (Table 4). Nevertheless, there were some notable differences between the growth conditions. There was a significant negative relation of LMA with the GAI for medium gap grown plants, but only a non-significant negative trend remained for large-gap plants (Table 2; Fig. 2). This was the result of a stronger increase in the LMA of the gapdependent species, when going from the medium to the large gap (Fig. 3). The chlorophyllLA was positively related to the GAI, a non-significant trend in the medium gap, but
a significant relation in the large gap (Table 2). Another difference with closed-canopy-grown plants was that carotenoidschl was negatively related to the GAI (Fig. 2), again a small effect in the medium gap, and a strong relationship in the large gap (Table 2). The magnitude of the effect of growth in the large gap relative to the closed canopy was used as a measure of plasticity for a particular trait. Factor analysis showed that, contrary to the trait values, the plasticity in the structural traits and strength had a positive association with the GAI (Fig. 1c), although not all individual relations were significant (Table 3; Fig. 3). Plasticity in ETR expressed on each basis showed no relationship with the GAI. There was a positive relationship in the case of plasticity in ETRLA in large-gap plants, but the maximum gap effect, found
13
Oecologia
Fig. 2 Mean leaf trait values plotted against the species’ GAI. Values for juveniles growing under a closed canopy (closed symbols) and in a large gap (open symbols). Bold regression lines denote significant regressions, thin lines denote non-significant regressions (P
Physiological ecology - Original research
Gap effects on leaf traits of tropical rainforest trees differing in juvenile light requirement Nico C. Houter · Thijs L. Pons
Received: 6 June 2013 / Accepted: 11 January 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract The relationships of 16 leaf traits and their plasticity with the dependence of tree species on gaps for regeneration (gap association index; GAI) were examined in a Neotropical rainforest. Young saplings of 24 species with varying GAI were grown under a closed canopy, in a medium-sized and in a large gap, thus capturing the full range of plasticity with respect to canopy openness. Structural, biomechanical, chemical and photosynthetic traits were measured. At the chloroplast level, the chlorophyll a/b ratio and plasticity in this variable were not related to the GAI. However, plasticity in total carotenoids per unit chlorophyll was larger in shade-tolerant species. At the leaf level, leaf mass per unit area (LMA) decreased with the GAI under the closed canopy and in the medium gap, but did not significantly decrease with the GAI in the large gap. This was a reflection of the larger plasticity in LMA and leaf thickness of gap-dependent species. The well-known opposite trends in LMA for adaptation and acclimation to high irradiance in evergreen tropical trees were thus not invariably found. Although leaf strength was dependent on LMA and thickness, plasticity in this trait was not related to the GAI. Photosynthetic capacity expressed on each basis increased with the GAI, but the large plasticity in these traits was not clearly related to the GAI. Although gap-dependent species tended
Communicated by Fernando Valladares. Electronic supplementary material The online version of this article (doi:10.1007/s00442-014-2887-9) contains supplementary material, which is available to authorized users. N. C. Houter · T. L. Pons (*) Department Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Padualaan 8, 3508 CH Utrecht, The Netherlands e-mail: [email protected]
to have a greater plasticity overall, as evident from a principle component analysis, leaf traits of gap-dependent species are thus not invariably more phenotypically plastic. Keywords Acclimation · Gap dependence · Leaf trait · Plasticity · Shade tolerance
Introduction Light availability is low under the canopy of undisturbed evergreen tropical rainforest. Creation of a gap, such as that caused by fallen trees, increases plant exposure to the sky. The resulting increased irradiance has a direct effect on plant growth and it also drives changes in other microclimatic conditions such as elevated daytime temperatures and associated vapour pressure deficit (Chazdon et al. 1996). Gap conditions can be conducive for plant growth as a result of the increased light availability but can also be stressful to plants. Species may need canopy gaps or large disturbances for establishment and further development; these are referred to as ‘gap-dependent species’. However, high irradiance, especially in combination with high temperatures, can be stressful for shade-tolerant species that are capable of establishing under a closed canopy (Mulkey and Pearcy 1992; Lovelock et al. 1994; Houter and Pons 2005). Gap-dependent species and shade-tolerant species are not clearly distinct functional groups. The requirement of gaps for regeneration is better considered as a continuum (Popma and Bongers 1988; Osunkoya et al. 1994; Poorter et al. 2004; Coste et al. 2005; Laurans et al. 2012). Several studies have shown that functional leaf traits vary systematically with a species’ light requirement for regeneration (Valladares and Niinemets 2008). Species from evergreen tropical rainforest that are strongly
13
Oecologia
dependent on gaps for regeneration generally have a low leaf mass per unit area (LMA). Their leaves have short life spans and they are not well defended against herbivores. They are further characterized by low strength and high photosynthetic capacity (Bongers and Popma 1990; Reich et al. 1992; Raaimakers et al. 1995; Coley and Barone 1996; Westoby et al. 2002; Poorter and Bongers 2006). The leaves of shade-tolerant species have opposite characteristics. This is evidence for a trade-off between investment in defence, that is favoured in shade-tolerants, and in photosynthetic functions, as favoured in gap-dependent species (Mooney and Gulmon 1982; Coley et al. 1985). Plants can acclimate to the variation in the light climate as found under a closed canopy and in gaps. Numerous studies indicate that at the leaf level, plants growing at high irradiance compared to their counterparts in the shade have thicker leaves with a higher LMA and an associated higher photosynthetic capacity per unit leaf area (Lambers et al. 2008; Poorter et al. 2009). These leaves are also stronger (Onoda et al. 2008). These increases with acclimation to high irradiance are opposite to the decrease in LMA and leaf strength with adaptation to gap conditions in evergreen tropical trees as mentioned above (Veneklaas and Poorter 1998; Lusk et al. 2008). The picture is different for photosynthetic capacity per unit leaf area, because both acclimation and adaptation to high irradiance lead to higher values (Kitajima 1994; Raai-makers). Several studies indicate that leaf-level plasticity associated with acclimation to high irradiance is larger for gap-dependent species compared to shade-tolerant ones (Strauss-Debenedetti and Bazzaz 1996; Ellsworth and Reich 1996; Valladares et al. 2000); but this is not invariably found for all traits (Popma et al. 1992; Rozendaal et al. 2006). Traits at the chloroplast level, such as photosynthetic capacity per unit chlorophyll and pigment composition, can also be modified with acclimation to the level of irradiance. Photosynthetic capacity per unit chlorophyll is higher in high-light compared to shade-grown leaves. This is the result of a change in the organization of the chloroplasts (Anderson et al. 1995; Hikosaka and Terashima 1995). Photon absorption is not a limiting factor for photosynthesis at high irradiance, which makes a relatively large investment in photosynthetic capacity efficient in these light conditions. However, in shade environments, photon absorption limits photosynthetic rates and shade-acclimated leaves typically have more chlorophyll relative to capacity (Evans 1989; Hikosaka and Terashima 1996; Pons and Anten 2004). The chlorophyll a/b ratio pertains to the relative investment in the light harvesting complex of photosystem II (LHCII), which is rich in chlorophyll b. A low chlorophyll a/b, as found in many shade-grown leaves, thus represents a large LHCII, which is consistent with the low photosynthetic capacity per unit chlorophyll. However, not all tropical plants exhibit a clear increase of chlorophyll a/b with increasing light availability
13
(Krause et al. 2001; Matsubara et al. 2009). Several carotenoids are associated with photoprotection in high-light conditions and their contents per unit chlorophyll tend to increase with increasing irradiance; this also occurs in tropical trees (Krause et al. 2004; Krause et al. 2012). Relationships of these chloroplast-level trait values and their plasticity with species’ juvenile light requirement has not been systematically investigated in tropical trees so far. The first question was to what extent leaf traits are associated with the regeneration niche of tropical rainforest trees and what the relationships between the traits are, with particular respect to the chloroplast-level traits that are not well studied in evergreen tropical trees? Tree species were selected that covered a wide range of requirements for canopy gaps for their regeneration. Trait values were related to an index of gap dependence of a species, which is equivalent to the inverse of shade tolerance. Young saplings of the trees were grown in common gardens in the forest under a closed canopy and in gaps of two different sizes. The shade-tolerant and gap-dependent species were thus exposed to an identical full range of irradiance from deep shade to almost full daylight as opposed to sampling from naturally occurring populations where the range is more limited and not necessarily identical. This enabled us to investigate the species under conditions of canopy openness where they are not easily found. Growth of the plants in the full range of irradiance allowed the comparison of full plasticity in the different traits and to address the second question: how is the plasticity of the traits associated with a species’ regeneration niche?
Materials and methods Study site The study was carried out in a largely undisturbed forest in Central Guyana at about 50 km south of the Mabura Hill township in the West-Pibiri compartment of the local timber concession (5°02′N, 58°37′W). The dominant soil is a Haplic Ferralsol, locally known as ‘brown sandy soil’. It is a highly weathered acidic soil that has a high iron, aluminium and manganese content, and is low in available nutrients (van Kekem et al. 1996). Average annual precipitation is around 2,700 mm (Pons and Helle 2011). Approximately circular gaps of different sizes had been created in the forest in 1996 (van Dam 2001; Houter and Pons 2005). A medium-sized and a large gap were used for this study. Plant material Young saplings of 24 species that are common in the area were used for the study. Ten of those are among the
Oecologia Table 1 Species used in the study, their family, and their gap association index (GAI) value
a
Ranges from 1 to 5, where low values indicate that regeneration from seed is predominantly found under a closed canopy and in small gaps and high numbers indicate predominantly regeneration in large gaps and clearings. Each value is the mean (± SD) of five estimates
Species
Family (subfamily)
GAIa
Oxandra asbeckii (Pulle) R.E. Fr.
Annonaceae
1.0 ± 0.0
Tapura guianensis
Dichapetalaceae
1.5 ± 0.6
Mora gongrijpii (Kleinh.) Sandw.
Leguminosae (Caesalpinioideae)
1.6 ± 0.9
Eschweileria sagotiana Miers
Lecythidiaceae
1.7 ± 0.6
Chlorocardium rodiei (Schomb.) Rohwer. Richter and v.d. Werff
Lauraceae
1.8 ± 0.8
Lecythis concertiflora (A.C. Sm.) Mori
Lecythidaceae
1.8 ± 0.4
Vouacapoua macropetala Sandw.
Leguminosae (Caesalpinioideae)
1.8 ± 1.3
Protium guianensis var. guianensis
Burseraceae
2.1 ± 1.3
Catostemma fragrans Benth.
Bombacaceae
2.3 ± 0.7
Licania heteromorpha Benth. var. perplexans Sandw.
Chrysobalanaceae
2.3 ± 1.3
Ormosia coccinea (Aubl.) Jacks.
Leguminosae (Papilionoideae)
2.5 ± 0.7
Dicymbe altsonii Sandw.
Leguminosae (Caesalpinioideae)
2.6 ± 0.8
Parinaria campestris
Chrysobalanaceae
2.7 ± 0.6
Pouteria speciosa
Sapotaceae
2.7 ± 1.2
Hymenea courbaril L. var. courbaril
Leguminosae (Caesalpinioideae)
3.1 ± 1.3
Pentaclethra macroloba (Willd.) Kuntze
Mimosoideae
3.2 ± 0.4
Carapa guianensis Aubl.
Meliaceae
3.3 ± 1.4
Inga spp.
Leguminosae (Mimosoideae)
3.4 ± 1.1
Peltogyne venosa (Vahl) Benth. subsp. densiflora (Spruce ex Benth.) M.F. Silva
Leguminosae (Caesalpinioideae)
3.8 ± 1.0
Sclerolobium guianense Benth. var. guianense
Leguminosae (Caesalpinioideae)
4.6 ± 0.4
Goupia glabra Aubl.
Celastraceae
4.7 ± 0.4
Laetia procera (Poepp.) Eichler
Flacourtiaceae
5.0 ± 0.0
Jacaranda copaia (Aubl.) D. Don subsp. copaia
Bignoniaceae
5.0 ± 0.0
Cecropia obtusa Tréc.
Moraceae
5.0 ± 0.0
hyperdominant species distinguished by ter Steege (2013) across Amazonia, and another eight are members of genera that contain one or more hyperdominant. The selected species differ in their requirement of canopy gaps for their regeneration. To what extent they depend on gaps was characterized for each species in a similar way to Osunkoya et al. (1994) and Mostacedo and Fredericksen (1999). The species were assigned to one of five categories by one of us (N. C. Houter), an experienced tree spotter (S. Roberts), and three ecologists with long-standing working experience in the local forest (P. van der Hout, H. ter Steege and R. C. Zagt). The categories were based on the occurrence of seedlings and small saplings in canopy gaps (1) mostly under a closed canopy and occasionally in small gaps, (2) mostly in small gaps and under a closed canopy, (3) mostly in gaps of different sizes and occasionally under a closed canopy, (4) exclusively in gaps of different sizes and not under a closed canopy of undisturbed forest, (5) typically in large gaps and clearings. The results were averaged and the means were used as an index of association of a species as juveniles with canopy gaps [the gap association index (GAI)]. Species and
their GAI are shown in Table 1. This index is thus representative for a species’ light requirement for regeneration and is inversely related to its shade-tolerance. Experimental design and leaf sampling The effect of growth of young saplings in gaps in the forest canopy on leaf trait values was investigated by comparing them with counterparts growing under the closed forest canopy. The objective was to measure the full range of plasticity of a species with respect to canopy openness. As most species typically do not cover the full range of canopy openness in their natural habitat, they were planted in these two conditions. Shade-tolerant species may not perform well in a large gap and may not exhibit the maximum (or minimum) trait values there as in gaps of smaller size. Hence, in addition to the control conditions under the closed canopy, seedlings were planted in a large gap (ca. 2400 m2) and in a medium-sized gap (ca. 320 m2). Four species, Tapura, Vouacapoua, Dicymbe and Inga, were not planted in the medium gap. This approach allowed us
13
Oecologia
to compare species under identical conditions in the full range of canopy openness from deep shade under a closed canopy to almost full daylight in a large gap. Our experimental approach is positioned between traditional shade house experiments, where plants are typically grown in pots under spectrally neutral screens, and sampling from populations under the gradient of canopy openness, where they naturally occur. Contrary to shade house experiments, our plants experienced the natural variation in irradiance, spectral, directional and temporal components of the light environment, and variation in other environmental factors associated with variation in canopy openness such as temperature, wind and humidity. Also possible pot effects are excluded (Poorter et al. 2012). Seeds were locally collected and germinated in a nursery (4.7 % of full daylight). When seeds were not available, seedlings were collected in the surrounding forest. An area of 130 m2 in the gaps was prepared by excavating roots and removing larger debris, and clearing secondary growth before planting. Ten plants per species were randomly planted at a spacing of 0.8 m. It was necessary to nurse sensitive species after planting in the high-light environment of the gaps, as indicated by preliminary tests. All plants were shaded for 1 to 2 months until growth had resumed and watered in dry periods to avoid excessive water stress. Litter from the surrounding forest was put on top of the soil and the plants received initially 5 g of slow-release fertilizer (Osmocote plus; release time 2–3 months). Daily photosynthetic irradiance was around 35 and 90 % of above-canopy values in the medium and large gaps, respectively, as measured with quantum sensors connected to a data logger (LI192 s and LI-1000; LI-COR, USA). The seedlings that were planted under the closed canopy received about 1 % of daily above-canopy photosynthetic irradiance. Plots with planted seedlings were fenced against larger herbivores. The gapdependent species (GAI > 4; Table 1) that did not survive in sufficient numbers in the deep shade conditions under the closed canopy of the primary forest were sampled in the most shaded places under a closed canopy in disturbed areas where relative irradiance was between 2 and 3 %. Their full range of growth conditions was thus slightly different from that of the more shade-tolerant species. The plants were grown in the gaps for about 5 months when new leaves had formed under the gap conditions at the time of sampling. We attempted to keep plant height equal between species at sampling, but some variation between species could not be avoided (24 ± 14 cm; mean ± SD) as a result of variation in seed mass (more than 5 orders of magnitude) and growth rate. It could also not be avoided that some plants growing under the closed canopy were smaller than gap-grown plants, particularly the small-seeded gap-dependent species. Cotyledons were fully exhausted at the time of sampling, hence, plants were beyond the seedling stage.
13
Recently matured leaves were measured for thickness, area, fresh and dry mass, toughness, pigments, nitrogen and the light-saturated electron transport rate (ETR). Sampling was done between March and May 2000 when daily photosynthetic irradiance was around 28 mol m−2 day−1. Photosynthetic traits Photosynthetic capacity was measured as the light-saturated ETR by means of chlorophyll fluorescence. A pulse-amplitude-modulation fluorometer (mini-PAM, Walz, Effeltrich, Germany) was used with the leaf clip holder (2030-B) for measurement of the quantum yield of PSII in the light (ΦPSII = ΔF/Fm’; where Fm’ is maximum fluorescence in light) and photon flux density (PFD). Measurements in the gaps were done in the morning before or shortly after direct sunlight hit the sample leaf. Additional lighting with a halogen lamp was used to increase irradiance to saturating levels when required. The intensity of the measuring light and the irradiance required for light saturation in each species and light condition was established in preliminary measurements. Irradiance was reduced when ΔF/Fm’ was lower than 0.2, which was achieved by adjusting the irradiance, for better accuracy of the ETR measurement. The saturating pulse was set at its maximum value (6 mmol m−2 s−1). ETR was calculated according to Genty et al. (1989) as ETR = PFD × ΦPSII × α × β, where ΦPSII = ΔF/Fm’. The leaf absorbance (α) was derived from chlorophyll per unit area (chl; μmol m−2) as α = chl/(chl + 76) (Evans and Poorter 2001), and an equal partitioning of photon absorption between the two photosystems was assumed (β = 0.5). ETR thus calculated provides a good measure of gross photosynthesis when cyclic electron transport is of minor importance (Genty et al. 1989). Structural, chemical and biomechanical traits Whole leaves or leaflets that had been used for the photosynthesis measurements were stored between moistened filter paper until further processing the same day. In the case of large leaves, such as those of Cecropia, about 100 cm2 of the leaf blades was used. Leaf area was measured with a leaf area meter (LI-1100; Li-COR) and fresh mass. Dry mass was measured after 3 days at 70 °C. Leaf mass per area (LMA) was calculated from the dry mass to area ratio. Thickness of the fresh leaf was measured with a thickness gauge (resolution 0.01 mm) excluding the veins. Pigments were extracted from three leaf discs (1.9 cm2) in 5 ml dimethylformamide after storage in darkness at room temperature for 4–10 days when extraction was completed. Absorbance was measured with a spectrophotometer (Shimadzu UV/VIS 120-01; Shimadzu, Kyoto), and chlorophyll a and b, and total carotenoids were calculated
Oecologia
according to Inskeep and Bloom (1985). Total leaf nitrogen per unit dry mass was measured on ground leaf material using an elemental analyser (Carlo Erba, Milan) on closedcanopy and large-gap plants only. Leaf strength was measured on separate but similar leaves to those used for the previous measurements according to Feeny (1970) and Choong et al. (1992). A leaf was put between two acrylic plates; the lower one had a hole of 5 mm diameter and the upper one a hole of 2 mm diameter. A freely moving metal rod (cross-section 2.5 mm2) with a beaker on top rested perpendicularly on the leaf. The force on the leaf was slowly increased by filling the beaker with water. The total weight when the rod penetrated the leaf was used to calculate the punch strength by dividing it by the cross-sectional area of the rod. Calculations and statistical analyses The effect of growth in a gap was expressed as the relative change in a trait value per species when comparing gap-grown plants (gap) with plants grown under the closed canopy (CC): (gap–CC)/CC. Two-way ANOVAs were used to evaluate species specific differences in gap effects on leaf trait values after log-transformation. The relationships with the GAI of the species mean trait values per growth condition and the gap effects on the traits were evaluated by means of regression analysis. Correlations between leaf traits per growth condition and between gap effects on traits were evaluated with the Spearman correlation coefficient (twosided). The maintenance of species ranking between growth conditions was calculated by pair-wise comparison of the species mean trait values and its significance was evaluated by calculating Kendall’s correlation coefficient. Associations among leaf traits per growth condition and plasticity of traits were analysed with factor analysis extracted from a correlation matrix using principle component analysis (PCA). Sampling adequacy and sphericity were tested (KMO and Bartlett tests), and solutions were orthogonally rotated (varimax rotation) and limited to a maximum of two factors. Plasticity per trait was calculated from the maximum (max.) and minimum (min.) mean values according to Valladares et al. (2006) as: (max.–min.)/max. The gap effect on a trait value and the plasticity index were highly correlated (mean r = 0.97). The statistical analyses were carried out using SPSS 20 (IBM, Armonk, NY).
Results Plants grown under a closed canopy The PCA plot shows that the photosynthetic variables were strongly associated with the GAI on the first axis
(Fig. 1a). This illustrates the highly significant increase in photosynthetic capacity (ETR) expressed on each basis with increasing gap dependence of a species (Fig. 2; Table 2). However, the leaf traits pertaining to strength and structure, punch strength, LMA, density and thickness, occupied a position more to the left on the first axis (Fig. 1a), as evidence of their negative relationship with the GAI (Fig. 2; Table 2) and with the photosynthetic variables (Supplemental Table S4). Punch strengthLA (for abbreviations see Table 2) was positively correlated with LMA and thickness, but not with density (Supplemental Table S4). ChlorophyllLA was not related to a species’ gap dependence in closed-canopy growth conditions; nor were the other pigment variables, chlorophyll a/b ratio and carotenoidschl (Table 2; Fig. 2). Due to the decrease in LMA with the GAI, chlorophyllDM increased with the GAI (Table 2). The same is true for chlorophyllN (Fig. 2), which is the result of a stronger increase with the GAI of chlorophyllDM compared to NDM (Table 2). Leaves of shade-tolerant trees compared to those of gap-dependent species growing in dense canopy shade were thus characterised by a low photosynthetic capacity expressed on each basis. Their leaves were strong and had a high LMA, and tended to be thicker and denser. Their chlorophyllLA, chlorophyll a/b and carotenoidschl were not different from those of gap-dependent species, but their chlorophyllDM and chlorophyllN were lower. Their leaves had also a somewhat lower NDM and their NLA tended to be somewhat higher. The effect of growth in gaps Growth in gaps compared to growth under a closed canopy had highly significant effects on all measured leaf traits as evident from a two-way ANOVA (Supplemental Table S1). The highly significant species × gap size interactions indicate that species responded differently to the canopy gaps. Most trait values increased as a result of growth in a gap, but some decreased, such as chlorophyll, expressed on each basis, and punch strengthDM (Table 3). Most trait values showed the largest difference when comparing closed-canopy with large-gap-grown plants. However, the gap effect had already reached its maximum in the medium gap for some traits such as LMA and ETRLA for some shade-tolerant species, and for carotenoidschl for gap-dependent species (Figs. 2, 3; Supplemental Tables S2, S3). The effect of growth in gaps is mainly discussed by comparing large-gap plants with closed-canopy plants, as not all traits were measured on medium-gap plants and not all species were included (Table 1). Medium-gap plants are considered where appropriate.
13
Oecologia
Fig. 1 Principal component analysis (PCA) of leaf traits and their plasticity in 23 species (all species except Pentaclethra). a–c Loadings of the leaf traits on the first two components that were extracted from the correlation matrix. d–f Species factor scores on the first component plotted against their gap association index (GAI). The GAI was excluded from the analysis in the latter case. Juveniles grown a, d under a closed canopy, and b, e in the large gap; c, f plasticity index for the large-gap and closed-canopy trait value comparison [(mean maximum value–mean minimum value)/mean maximum
value]. a–c Variance explained by each of the components in parentheses (for axis 2 above the axis). The second percentage refers to the PCA without the GAI. Dry mass-based traits were excluded where leaf area-based traits were available. Also, chlorophyll a/b was not included as it was not related to the GAI and reduced the explained variance of the model. thick Thickness, dens density, strength punch strength, chl chlorophyll, carot carotenoidschl, A light-saturated electron transport rate, Tg Tapura, Co Cecropia
There was a similar clustering in the PCA plot of the large-gap compared to closed-canopy-grown plants (Fig. 1a, b) and regressions of trait values on the GAI showed also several similarities (Table 2). The similarity is also expressed in the maintenance of the species ranking in the traits for large gap compared to closed-canopy-grown plants (Table 4). Nevertheless, there were some notable differences between the growth conditions. There was a significant negative relation of LMA with the GAI for medium gap grown plants, but only a non-significant negative trend remained for large-gap plants (Table 2; Fig. 2). This was the result of a stronger increase in the LMA of the gapdependent species, when going from the medium to the large gap (Fig. 3). The chlorophyllLA was positively related to the GAI, a non-significant trend in the medium gap, but
a significant relation in the large gap (Table 2). Another difference with closed-canopy-grown plants was that carotenoidschl was negatively related to the GAI (Fig. 2), again a small effect in the medium gap, and a strong relationship in the large gap (Table 2). The magnitude of the effect of growth in the large gap relative to the closed canopy was used as a measure of plasticity for a particular trait. Factor analysis showed that, contrary to the trait values, the plasticity in the structural traits and strength had a positive association with the GAI (Fig. 1c), although not all individual relations were significant (Table 3; Fig. 3). Plasticity in ETR expressed on each basis showed no relationship with the GAI. There was a positive relationship in the case of plasticity in ETRLA in large-gap plants, but the maximum gap effect, found
13
Oecologia
Fig. 2 Mean leaf trait values plotted against the species’ GAI. Values for juveniles growing under a closed canopy (closed symbols) and in a large gap (open symbols). Bold regression lines denote significant regressions, thin lines denote non-significant regressions (P
