Applied tree ecophysiology is focused on the study of optimal environmental or habitat requirements to improve the success of tropical tree plantations and forest restoration programs. In past decades, strategies development for endangered plant species management using ecophysiological techniques under nursery conditions, has become a center of interest for many conservationists, because of its low costs, great amount of replicates that can be held in a little space, and precise results that describe response of tree species to different factors (Cooke et al., 2013; Corea, Arnaez, Moreira, Cordero, & Castillo, 2014; Guzmán & Cordero, 2013).

When looking to the habitat requirements of tree species, there are many resources involved to determine optimal conditions for each species, such as the water availability (Comita & Engelbrecht, 2009), nutrient supply (Walters & Reich, 2000), and soil conditions (Passioura, 2002). However in tropical forests, light requirements have been considered as the major environmental factor influencing the establishment, growth, development and reproduction of many tree species (Lambers, Chapin, & Pons, 2008). Presently, response of growth and photosynthetic traits of different endangered species when planted in either shade or exposed sites must be known in order to improve successful reforestation practices and conservation strategies (Aleric & Kirkman, 2005; Smith, Wu, & Green, 1993). To accomplish this, the use of artificial light treatments under nursery conditions can be useful to evaluate optimal habitat conditions for endangered species. In addition, population conditions of these species are typically in such a reduced condition, that field and community surveys are not enough to precisely extrapolate their habitat requirements.

Some photosynthetic and morphological traits of endangered species stand out as key features to classify their light preferences, mostly because they are plastic traits that respond multidimensionally to several light regimes, and whose variation trends under natural light environments can be inferred (Valladares & Niinemets, 2008). For example, it is a general trend that rates of carbon assimilation and specific leaf area under nursery conditions of shade-intolerant and tolerant species vary accordingly with the behavior found under natural conditions (Aleric & Kirkman, 2005). However, those trends in growth and survival rates of species under natural conditions are not necessarily similar as under controlled conditions, because these depend on whole habitat features. These kind of responses have led to consider that physiological and morphological traits are entities with a multidimensional response to light preferences.

The present study was conducted to analyze the effects of light regimes on Cedrela salvadorensis Standl. seedlings under nursery conditions by considering a set of morphological, biomass relations and detailed gas exchange variables. In Costa Rica, C. salvadorensis is critically endangered (MINAE Decree Nº 25700, 1997) and their distribution is very limited, because it has a high habitat reduction (74 %) with a high rate of exploitation (Estrada-Chavarria, Rodriguez-Gonzales, & Sanchez-Gonzales, 2005). Because of this, this study focused on the ecophysiological response of this species to different light environments, and how this response could have implications in programs of reforestation or reintroduction where this species is involved. Two main central questions were addressed: (1) How does C. salvadorensis adjust photosynthetic performance, growth, and biomass to different light environments? and (2) Is there a multidimensional response of biomass allocation and foliar physiology to different light regimes?. These two questions were responded through a biomass allocation analysis along with leaf photosynthesis changes due to light response and internal CO2 response curves. More than an ecophysiological perspective, we wanted to enhance and interpret the results to exalt and recommend procedures for optimizing seedling establishment and growth of this species in forest restoration programs.

MATERIALS AND METHODS

Study species: Work was done with seedlings of the neotropical endangered tree Cedrela salvadorensis Standl (Meliaceae), a tree species distributed from Mexico to Panama between 0 to 1 000 masl. In Costa Rica, the species is found in wet and dry deciduous and semi-deciduous forests, where a large wood extraction and limited natural regeneration have occurred (Jiménez, 1999); for this reason, the Costa Rican government proclaimed an executive decree declaring this species as forbidden for extraction.

Study site and experimental design: We conducted this study at the Instituto de Investigación y Servicios Forestales of the Universidad Nacional de Costa Rica (10º 01’ 20’’N - 84º 06’ 36’’, 1 270 masl), from June 21 to November 4, 2011. We exposed forty seedlings of C. salvadorensis to four light treatments (10 for each treatment) created with superimposed neutral shade cloth: 75, 45, 15 y 3.5 % of light, respect to full sun exposure. We determined the light conditions by measuring photosynthetic photon flux density (PPDF, µmol/m2.s) with a quantum sensor during short morning periods, which presented the maximum PPDF values of the studied season. Seedlings were obtained from seeds collected from five mother trees dispersed in a seasonal deciduous forests located at Valle del Sol, Southwest area of Costa Rica Central Valley. Seeds were germinated using fine river sand on the top of long concrete tables (10 m long x 1 m side x 10 cm depth) inside a greenhouse. Sand was carefully washed and sieved in a 2.0 mm mesh size sieve. Seedlings were transferred to plastic bags of 1 647 cm3 filled with a Typic distrandept soil (US Department of Agriculture, 1982) taken from the A horizon of nearby coffee plantations and mixed with sand (3:1 soil to sand). We maintained the seedlings under nursery conditions at 78 % shade for a period of six months, until they were used for this experiment.

Each treatment consisted of five pairs of seedlings from five different mother trees. The placement of individuals within each treatment followed a Latin Square Design with occasional rotation. After treatment set up, we monitored the acclimation progress following plant growth by measuring plant height and recording the presence of new leaves on June 21, August 18, October 06 and November 4 2011. To avoid water and nutritional deficiencies, the plants were kept moist every day with an automatic watering system, and plants were fertilized with four grams of complete commercial low-release (5-6 months) fertilizer (Osmocote Plus, 15-09-12 with minor elements). After the 135-day acclimation period, leaf physiological traits were measured by gas exchange analysis from light-response and CO2 response curves, using a Photosynthesis Systems Li6400XT (LiCor Inc, Nebraska, U.S.A.). Final biomass and allocation variables were measured after harvesting.

Biomass allocations: We harvested each plant to measure the total fresh leaf area (LA, cm2) with a leaf area meter (Li-3100, LICOR Inc., USA), and then we dried the harvested plants at 58 °C for more than 72 hours to measure the root (R, g), shoot (S, g), total dry biomass (TB, g), and total leaf dry biomass (LB, g). From this, we calculated the following ratios: root to shoot ratio (R/S), specific leaf area (SLA, as LA/LB, cm2/g), leaf area ratio (LAR, as LA/TB, cm2/g), leaf mass ratio (LMR, as LB/TB), shoot mass ratio (SMR, as S/TB) and root mass ratio (RMR, as R/TB) (Lambers et al., 2008).

Gas exchange: We selected four plants per treatment to measure physiological characteristics through leaf gas exchange analysis. Measurements were made using a portable photosynthesis system LI-6400 (LI-COR, USA), open configured, equipped with an artificial light source and CO2 assembly, on mature and fully expanded leaves. We performed light and CO2 response curve following Tambussi and Graciano (2010). Leaf temperature was maintained between 25-27 ºC and relative humidity between 65-80 % for both kinds of curves. Light response curves were made at 385-395 ppm CO2 concentration. We measured the carbon assimilation uptake at several irradiances ranging from 1 500 to 0 µmol/m2s by pre-illuminating leaves at 1 250 µmol/m2s1 for 2-3 min. Carbon assimilation (A, µmolCO2/m2s) versus irradiance (I, µmol/m2s) data was fitted by a non-rectangular hyperbola model (Marshall & Biscoe, 1980; Thornley & Johnson, 1990):

where A is rate of carbon assimilation (µmolCO2/m2s), Rdark is the dark respiration rate (µmolCO2/m2s), Qapp is apparent quantum yield (mol/mol), I is the applied photosynthetic photon flux density (µmol/m2s), Amax is the maximum assimilation rate (µmolCO2/m2s) and Ө describes the curvature. This model was fit by nls2 package (Grothendieck, 2014) of R software (R Development Core Team, 2014). All fitted curves showed r2 values greater than 0.95. Likewise, we calculated independently the light compensation point (Ic, µmol/m2s) by interpolation at “x” line interception of linear regression analysis of data points between 0 and 60 µmol/m2s. Because the values of Amax were presented close to values of light saturation of 1 000 µmol/m2s, we extracted from irradiances of 1 000 µmol/m2s the stomatal conductance (g, mmol H2O/m2s), transpiration (E, mmolH2O/m2s), and CO2 assimilation (A1000), and then we estimated the water use efficiency (WUE, µmolCO2/m2s/mmolH2O/m2s) by the ratio of A1 000/E.

We carried out a CO2 response curve after the light response curve in the same leaf, varying CO2 concentration between 50-900 ppm at a light irradiance of 1 000 µmol/m2s and atmospheric pressure of 86-87 Pa. We fitted the A versus internal carbon concentration (Ci) data following Ethier and Livingston (2004) version of Farquhar’s model (Farquhar, von Caemmerer, & Berry, 1980) to extract different leaf biochemical parameters: maximum rate of carboxylation (Vcmax, µmolCO2/m2s), maximum photosynthetic electron flow rate (Jmax, µmole-/m2s), day respiration rate (Rday, µmolCO2/m2s), chloroplast CO2 compensation point (Γ, µmol/mol) and mesophyll conductance (gm, molCO2/m2s).

We applied three mixed design of analysis of variance to determine the effects of time, treatments, and their interaction on the height, amount of leaves and amount of new leaves. Also we compared the treatments effects on the biomass allocation, light response and A-Ci response curves parameters by analysis of variance (ANOVA). We performed simple linear regressions to show the biochemical effects of Vcmax, Jmax and Γ on Amax. We also performed two principal component analyses (PCA) from a correlation matrix of biomass allocation and leaf gas exchange parameters to evidence differential weight on the total value variation. From the first principal component of each PCA, an analysis of covariance (ANCOVA) was performed of the first components of biomass allocations and leaf gas exchange factor scores as continuous variables using the light treatments as categorical factor, used as an indirect test of a multivariate response due to the light treatments. We conducted the previous analyses using R software version 3.1.1 (R Development Core Team, 2014).

RESULTS

Growth and biomass allocations: Throughout the 135 days of study, all plants showed increases in height (Fig. 1a), where plants at 75 % light treatment showed more increases than other treatments. Independently of treatment, plants presented reductions in the stem height increase by the end of the period, along with a final reduction of the amount of leaves and new leaves were produced (Fig. 1b and Fig. 1c). At day 58, plants increased the amount of leaves by 2.5±1.69 leaves, but plants decreased this mean to 1.58±1.92 leaves compared to baseline. However, lower light treatment presented plants with more leaves than other treatments. In the same way, the number of new leaves produced after 58 days was higher (2.95±1.47 leaves) than at the end of the study (0.51±1.71 leaves). Mixed design ANOVA showed a significant temporal effects on plant height and amount of leaves and new leaves, with no significant treatments*time interaction effect (Table 1). Only in the amount of leaves, the mixed design ANOVA showed a significant treatment effect.

Comparisons of four light treatments evidenced that there is a strong light influence on some biomass allocations parameters (Table 2), where TB, SLA, LAR and LMR showed significant differences between light treatments; meanwhile, the lighter treatments of 75 and 45 % presented greater TB and lower SLA, LAR, LMR values. SLA, LAR and LMR increased and TB decreased with decreasing irradiance, respectively. SMR, RMR, and R/S showed no significant light effect between treatments.

Gas exchange: Only Amax presented significant differences between light treatments in the A-I response curve (Table 3). Highest and lowest values of Amax were measured in 45 % and 3.5 % light treatments, respectively. All other parameters extracted from light response curves presented slightly higher values in lighter treatments, with no significant effects between them. Qapp showed a non-significant inverse trend. The A−Ci response curve parameters differ significantly between treatments (Table 3), except Rday. Highest values of Vcmax, Jmax and gm, and lowers values of Γ were found in 45 % light treatments. Linear relationships (Fig. 2) evidenced significant responses of Amax to biochemical leaf properties, showing high values of Vcmax and Jmax and lower values of Γ associated with high rates of assimilation.

Whole-plant response: PCA statistics for growth and biomass allocation (Fig. 3a) indicated that more than 66 % of variation was explained by the first two components. The first component is mostly influenced by LAR, LMR and SLA (eigenvector= 0.50, 0.42 and 0.41, respectively), and the second component by SMR, RMR, and SLA (eigenvector= -0.69, 0.63 and 0.26, respectively). For the foliar physiology variables, PCA explained 67 percent of the variation (Fig. 3b), where the first component is mostly influenced by Vcmax, Jmax and Amax (eigenvector= 0.42, 0.41 and 0.38, respectively) and the second component by Γ, Rday and WUE (eigenvector=-0.46, 0.35 and 0.33, respectively). Linear relationship between the first principal components from the two PCA showed that high leaf physiological scores were related with low scores of biomass allocations (Fig. 3c). ANCOVA comparisons showed that the biomass allocation traits were affected by light treatments (F3,16= 7.96; P < 0.001) and foliar physiology (F1,16= 20.10, P < 0.001), without interaction between these (F3,16= 0.75; P > 0.05), suggesting the existence of a multivariate response.

DISCUSSION

Results of monitoring leaf production showed a strong light-dependent acclimation of C. salvadorensis to light treatments. Although this study was conducted during a shorter period than a year, the responses of biomass allocation and gas exchange suggested a quick acclimation of this species to light regimes. During the monitoring, no differences were found in height, number and production of leaves within each treatment, but a reduction in the production and the number of leaves. Surprisingly during the early stage of development of these seedlings, the reduction in the production and number of leaves presented, could be associated to the inherent phenological patterns of the species, which is described as a deciduous species from November to March (Corea et al., 2014; Rojas-Rodríguez & Torres-Cordoba, 2013). This latter could have major implications in the reforestation programs, so according to the plant phenology, we recommend three practices for the transplantation and reforestation: i) keep the areas of reforestation within the premontane deciduous and semideciduous forests with long dry season (4-6 months), ii) avoid dry season during their early leaf loss, and iii) avoid dimer habitats. These types of practices are highly recommended for deciduous and other tropical tree species in some studies (Elliott et al., 2003; Wishnie et al., 2007), because dry sites can reduce significantly the growth rates, survival and acclimation of the species, especially in seedlings stage.

The continuous monitoring of seedlings of C. salvadorensis to different light conditions in terms of plant height, leaf number and production, might not be effective indicators of the acclimation of the species to different light environments, at least during the short period investigated, contrary of what happened with variables from foliar physiology and biomass allocation. In general, changes in the size and the amount of leaf in trees have been associated in long-term studies where the environmental conditions like drought or light regimen can have strong repercussions on the increases or decreases of these traits during the growth (Wishnie et al., 2007). However, in short-term studies like this, changes in the biomass allocation and gas exchange due to light availability in traits like SLA and Amax are related to the direct advantage for light interception in higher or low-light environment (Poorter, 1999, 2001), where this interception have strong implications on the plant performance and the assignment of structures to capture resources.

General observations from the results indicated that the biomass allocation had a great weight in the acclimation of this species to contrasting light regimes, especially the biomass allocation in leaf traits. Some studies showed that leaf characteristics such as higher SLA may allow seedlings to harvest light more effectively at low irradiance (Loach, 1970) which can maximize the carbon gain per unit leaf mass (Evans & Poorter, 2001). However, the higher SLA and LMR in C. salvadorensis in the treatment with low light could be associated with a small growth or lower biomass accumulation in a dim light environment. Although variables such as R/S, SMR and RMR apparently did not respond to light treatments, it is clear that these variables are important in the balance between investment in light interception and water and nutrient uptake by organs (Poorter, 2001). Therefore, we do not discard that these variables in long-term studies and with other environmental factors also play an important role in the acclimation or survival of the species to different light regimes, or they will become important when planted under field conditions.

From a general perspective, we considered that the changes in the biomass allocation to high or dim light environments in C. salvadorensis, can be related to an economic advantage to exploit and efficient use of the light resource according to its availability. In this sense it is likely that the observed changes in the biomass allocation are related to resource-conservative and resource-acquisitive strategies (Adler, Fajardo, Kleinhesselink, & Kraft, 2013). For example, in this study, increases in the LMR in the low light treatments could be associated with resource-conservative strategy for the exploration of light in a low light environment (Givnish, 1988; Loach, 1970). Likewise, increases in SLA in the high light treatments could be associated with resource-acquisitive strategy to harvest more light per area in high light environments (Adler et al., 2013). We suggest that if the adaptation in the biomass allocation respond to conservative-acquisitive strategies in face to different light environments, it could prevent the environmental filtering of high or dim light environment and it could promote their success in the forest.

When considering the gas exchange performance, the differences between the biochemical characteristics of leaf physiology to light treatments, evidenced a joint response of leaf biochemical capacity and Amax. In general, higher Amax values found in treatments with higher irradiance, showed typical responses of light-demanding species (Schaedle, 1975); however, the responses to the light treatment observed in this species can have different explanations. For example, studies suggest that the resistance in the molecules (CO2 and H2O) transport inside and outside leaf mesophyll, can limit different physiological processes (Medrano, Escalona, Bota, Gulias, & Flexas, 2002), for this, it is possible that conductance effects like g and in particular the lower values of gi in the 3.5 % light treatment could be the responsible for the differences in Amax, Vcmax, Jmax, and Γ. Likewise, the differences in these parameters could suggest that the assimilation capacity, and the biochemical ability of carboxylation and electron flow exhibit symptoms of photosynthetic stress like photoinhibition. In many cases, differences in the assimilation, carboxylation and electron flow in leaves are the result of environmental stress like a high radiation or high temperature, and they will have strong implications in other processes like respiration or the light compensation point (Long, Humphries, & Falkowski, 1994; Niinemets, Kull, & Tenhunen, 1998). Either by changes in the leaf conductance or environmental triggers which induce a physiological stress, here the changes in Amax, Vcmax and Jmax suggest that C. salvadorensis presents a greater capacity of gas exchange in 45 % light treatments. From an ecological perspective, it is possible that these results obtained at the 45 % shade treatment seems to coincide with light regime perceived inside natural large gaps (>400 m2) in tropical forests (Chazdon & Fetcher, 1984).

The multidimensional response found evidences that light acclimation of the species is directly related to concomitant changes in leaf gas exchange physiology and biomass allocation patterns (Bazzaz & Pickett, 1980; Pearcy, 2000). In this sense, it is likely that precise monitoring of single physiological or biomass allocation traits may help to follow a species light acclimation trend. For example, changes in plant LAR, LMR or SLA in response to different ranges of natural conditions could prove to be indicative of the gas exchange performance of the plant, which can provide a simple protocol for monitoring of the species under different conditions. Likewise, although the degree of phenotypic plasticity of this species to light treatments was not measured, it is possible that the great variations of their trait in terms of biomass allocation and gas exchange could be indicators of the great phenotypic plasticity induced by changes in the light regimens as shade-intolerant species. This suggestion has been clearly demonstrated in this kind of species and associated with changes in SLA, Amax, and LAR (Rozendaal, Hurtado, & Poorter, 2006; Valladares, Martinez-Ferri, Balaguer, Perez-Corona, & Manrique, 2000).

There is a strong light-dependent acclimation of C. salvadorensis to light regimes, where the overall performance of this species under nursery conditions seems to be optimized at light levels corresponding to 45 % shade environment. This light condition is easy to obtain with low cost and low time-demanding practices in a simple and rustic nursery or in natural conditions like forest edge, agroforestry systems with low density of trees, or secondary forests. Caution must be taken to extrapolate these results to open field conditions in a greater scale initiative, where water availability and fertilization activities may be reduced. With this in mind, the use of this species in any forest restoration programs has to be more careful in the selection of sites with suitable light conditions for the growth and establishment of species, such as avoiding dry season during their early leaf loss and avoiding lighter and dimer habitats. Any effort in this sense will help reduce the speed of biodiversity genetic loss of this neotropical endangered tree species. Future work must be directed to demonstrate to what extent these acclimation responses may change during field establishment under natural or plantations systems, and how these responses can allow the survival of this species in the highly diverse tropical forests.

ACKNOWLEDGMENTS

We thank Maricruz Torres, Leonel Orozco and Jairo Hidalgo for their assistance with the plants and data collection. Thanks to Elmer García who provided valuable comments and recommendations on the manuscript. This study was supported by two grants from the Fondo Especial para la Educación Superior through the (Consejo Nacional de Rectores de Costa Rica), one to Roberto Cordero and other equipment grant (Licor-6400XT system) to Escuela de Ciencias Agrarias, both to Universidad Nacional of Costa Rica.

RESUMEN

Asignación de biomasa e intercambio de gases son afectados por las condiciones de luz en plántulas de Cedrela salvadorensis (Meliaceae) en peligro de extinción. La determinación de las condiciones lumínicas favorables por especie y estado de vida es trascendental para las estrategias de conservación ex situ y in situ de especies de árboles en peligro de extinción, y su utilización como plantaciones forestales. Esto se vuelve especialmente importante cuando el material de siembra es escaso. Aquí, nosotros estudiamos las respuestas en asignación de biomasa y en intercambio de gases a luz y CO2 en plántulas de Cedrela salvadorensis crecidas bajo condiciones lumínicas similares a las que esta especie enfrenta en la naturaleza. Durante 135 días, grupos de diez plántulas fueron colocadas bajo condiciones de 75, 45, 15 y 3.5 % de exposición total al sol obtenidas por medio de sarán bajo condiciones de vivero. Una serie de variables de asignación de biomasa y parámetros de intercambio de gases (fotosíntesis según curvas de respuesta a la luz y a la concentración de carbono interno) fueron medidas al final del periodo de crecimiento en las plantas de los cuatro tratamientos. De acuerdo con los análisis de componentes principales, valores altos de respuesta de intercambio de gases están asociados con valores bajos de rasgos de asignación de biomasa. Los cambios observados se encuentran asociados con las estrategias recurso-conservativas y recurso-adquisitivas donde C. salvadorensis aclimata sus rasgos para la exploración y explotación de luz en ambientes con escasa o excesiva radiación lumínica, respectivamente. Los análisis multivariados muestran también que las plantas tienen un alto rendimiento a 45 % de luz ambiental. Estos resultados sugieren que el 45 % de ambiente lumínico es el hábitat lumínico óptimo de esta especie en el estado de desarrollo estudiado. Nuestros resultados tienen importantes implicaciones para escoger el mejor hábitat natural para un exitoso establecimiento de C. salvadorensis. Por esto, proponemos consideraciones prácticas para programas de reforestación y reintroducción donde esta especie estaría involucrada.

Palabras clave: curvas de respuesta al CO2, árboles en peligro de extinción, preferencias lumínicas, curvas de respuesta a la luz, árboles tropicales.

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