Cacao (Theobroma cacao L.) has traditionally been considered a crop that requires shade, and consequently it is implemented under agroforestry systems (AFs) in order to regulate the level of incident solar radiation. However, optimal shade levels for this tree crop may vary depending on the climate conditions of where it is grown. Here we analyzed the physiological performance of cacao under three different AFs in the Colombian Amazon that differed in solar radiation patterns: high (HPAR), medium (MPAR), or low (LPAR) mean daily incident radiation. The physiological performance was characterized using photosynthetic variables in leaves such as light- and CO2-response curves, chlorophyll a fluorescence parameters, and total chlorophyll and carotenoid contents, in conjunction with other leaf functional traits. Cacao trees exposed to HPAR showed an improved physiological performance as compared to those from the other two AFs. Compared to MPAR and LPAR, cacao trees in HPAR doubled the rate of net carbon assimilation and reached higher maximum rates of RuBisCO carboxylation and RuBP regeneration. Moreover, cacao trees in HPAR presented photoprotection mechanisms that avoided photoinhibition, which was accompanied by a greater non-photochemical quenching coefficient and biochemical and morphological adjustments (low chlorophyll but higher carotenoid contents and low specific leaf area) compared to cacao trees from the other AFs. Overall, our results show that, due to the high cloud cover in the Colombian Amazon, cacao plantations under conditions of sparse shade maximized their carbon use, showing an improved physiological performance as a result of higher photosynthetic rates and energy dissipation mechanisms. If the crop were managed with sparse shade, the paradigm that favors the cultivation of cacao under shade would be called into question in the Colombian Amazon and other regions with similar climatic conditions.
Citation: Suárez Salazar JC, Melgarejo LM, Casanoves F, Di Rienzo JA, DaMatta FM, Armas C (2018) Photosynthesis limitations in cacao leaves under different agroforestry systems in the Colombian Amazon. PLoS ONE 13(11): e0206149. https://doi.org/10.1371/journal.pone.0206149
Editor: Maya Dimova Lambreva, National Research Council of Italy, ITALY
Received: April 29, 2018; Accepted: October 8, 2018; Published: November 1, 2018
Copyright: © 2018 Suárez Salazar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was conducted with the financial collaboration of COLCIENCIAS (http://www.colciencias.gov.co/) using BID funds (Inter-American Development Bank) under the framework of the project "Characterization and typification of traditional plantations of Theobroma cacao L. as a strategy for the design and implementation of agroforestry systems in the Bajo Caguan (Cartagena del Chaira)," implemented by Universidad de la Amazonía (Colombia) under the special contract 590 of 2010 to JCSS, as well as using Doctoral Training funds from Amazon University under the stamp "Pro-development of Amazon University" Law No. 1301 of 2009. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cacao (Theobroma cacao L.), or cocoa, is one of the most important perennial crops in the world . It is native to the Amazon rainforest , specifically the Colombian-Brazilian region straddling the Caquetá River , and has developed under high levels of precipitation (1,500–2,000 mm) and understory low light conditions . The species shows photosynthetic characteristics of a shade tolerant species, exhibiting saturated rates of net carbon assimilation (A) at photosynthetically active radiation (PAR) levels between 200 and 750 μmol m-2 s-1 [5–7], as well as a low light compensation point (LCP) between 5 and 57 μmol m-2 s-1 [5, 7, 8] and a maximum A within the range of 1 and 8 μmol m-2 s-1 [5–7, 9]. For these reasons, cacao crops around the world are usually established in shady environments [10, 11].
Solar radiation and sunshine (daylight hours) vary greatly across the cacao-growing regions of Colombia . For this reason, it is suggested that the structure (spatial distribution) and composition of cacao agroforestry systems (AFs) should be adjusted to generate an adequate level of shade for the cacao crop due to its sensitivity to high solar radiation inputs [11, 13, 14]. In parallel, the use of shelter trees that give shade to cacao plantations can modify the microclimatic conditions (e.g., temperature, humidity, vapor pressure deficit), which can potentially affect cacao’s stomatal conductance, transpiration rates, and A  as well as its agronomic performance .
Cloudy days prevail in the Colombian Amazon. Even on these days, average values of solar radiation of 766 μmol m-2 s-1, mean temperature of 22 °C, and humidity of 85% have been reported . Due to these prevailing environmental conditions, there is some uncertainty on the real need to establish cacao plantations under shade conditions (AFs). In this regard, we previously evaluated cacao crops under AFs that are typical of the Colombian Amazon; we characterized the species composition, canopy structure and light availability in these AFs, which differ in degree of shade according to the shelter species that accompany the cacao plant, as well as the density of these species  (see Materials and methods). In this present study we analyzed the effect of transmitted radiation levels on the physiological performance of cacao plants by measuring chlorophyll (Chl) a fluorescence parameters and photosynthetic traits in leaves. We expected that the optimal physiological behavior of cacao would occur in the AFs with the greatest shade and that receive the least radiation due to a high density and diversity of accompanying species (the Diversified multistrata or LPAR treatment, which receives a mean daily PAR transmission levels of 28%; PAR = 300 μmol m-2 s-1). These PAR values are within the optimal values for cacao crops, as reported by other authors [5, 6, 11]. Proper management of the cacao crop within AFs could translate into improvements in photosynthetic rates given that a decrease in solar radiation loads may improve the microclimatic conditions for the cacao plants, thus allowing greater leaf gas exchange rates and ultimately improving the cacao agronomic performance [14, 18, 19].
Materials and methods
Study area, agroforestry systems and sampling procedures
Measurements were taken for cacao plants under different AFs at the Macagual Center for Investigation—Amazon University (1°37’N and 75°36’W at 360 m a.s.l.), Colombia. The climate is warm-humid, characteristic of the ecosystem of tropical rainforests, with a mean annual precipitation of 3,800 mm, 1,200 sunshine hours year-1, a mean temperature of 25.5°C, and a relative humidity of 84%. Cacao plants were planted in October 2012 in a regular pattern with a distance among plants of 3 m in three plots or AFs of 25x50 m each, and with a total of 138 cacao plants per plot. The genotype used was the cacao clone CCN51 which was grafted onto plants of the IMC67 genotype, which was previously germinated in a nursery. After 5 months of grafting, the cacao saplings were planted in the experimental area. Each sapling was fertilized with one dose of 46 g of urea, 18 g of di-ammonium phosphate (DAP), and 279 g of K2SO4. All AFs plantations were established in a flat area with similar soil characteristics: soil had a clay-loamy texture with a bulk density that ranged between 1 and 1.3 g cm-3, a mean organic carbon content of 1.35%, mean pH of 5.5, available P content (Bray- II) of 2.58 mg kg-1, saturation of total bases of 7.1% (Ca: 0.38 meq 100g-1, Mg: 0.1 meq 100g-1, K: 0.14 meq 100g-1, Na: 0.1 meq 100g-1, total bases: 0.8 meq 100g-1), a cation exchange capacity of 11.3 cmol(-) kg-1, and an exchangeable aluminum content of 6.3 cmol(+) kg-1 with a percentage of Al saturation of 73.4%. Although the cacao plantation (cacaotal) was identical in the three plots, each AF differed from each other based on the type and density of the accompanying shelter species (henceforth called vegetation), that were planted in 2008 in rows with a North-South orientation and that created an upper canopy with varying levels of shade. Two of the AFs included accompanying timber species (Cariniana pyriformis, Calycophyllum spruceanum) and a third AF included Musaceae species (plantain; Musa paradisiaca). The three cacao AFs compared in this study were as follows: i. cacaotal with Musaceae plantation and high mean daily incident radiation (HPAR) with the plantain trees planted at a density of 527 trees ha-1 (i.e., one tree per 6 x 3 m area) allowing a daily mean transmitted PAR of 700 μmol m-2 s-1 (with mean midday values of 1,300 μmol m-2 s-1); ii. low diversity of clustered vegetation and medium mean daily incident radiation (MPAR) with the average shade generated by clusters of trees with thin crowns (Cariniana pyriformis, Calycophyllum spruceanum) allowing a daily mean transmitted PAR of 400 μmol m-2 s-1 (with mean midday values of 900 μmol m-2 s-1), and a density of 35 trees ha-1 (i.e., one tree per 12 x 25 m area); iii. diversified multistrata vegetation and low mean daily incident radiation (LPAR) with intense shade (Cariniana pyriformis, Calycophyllum spruceanum) allowing a daily mean transmitted PAR of 300 μmol m-2 s-1 (and mean midday values of 500 μmol m-2 s-1), and a density of 55 trees ha-1 (i.e., one tree per 12 x 15 m area). Macro-environmental factors (general climate and soil) remained constant for all plots. Due to experimental restrictions, the AFs were created in large plots and were not repeated. All of the measurements were performed in four cacao plants per AFs that were randomly chosen. Because of the clonal and coetaneous nature of the plants, it is assumed that differences recorded at the physiological level between systems are due to the effect of the AFs on the micro-environmental variables under the canopy, as well as to the effects of these variables on the physiological behavior of cacao, i.e. in the absence of confounding factors. The PAR was measured every 10 min with a quantum sensor (SQ-420, Apogee Instruments, Logan, UT, USA) located three meters high and installed with a WatchDog 2900ET weather station (Spectrum Technologies, Inc., Texas, USA) for each AF for 180 days; from here, we recorded the mean hourly PAR during daylight hours (Fig 1).
The agroforestry systems for cacao were: cacaotal with Musaceae plantation and high mean daily incident radiation (700 μmol m-2 s-1; HPAR), low diversity of clustered vegetation and medium mean daily incident radiation (400 μmol m-2 s-1; MPAR), and diversified multistrata vegetation and low mean daily incident radiation (300 μmol m-2 s-1; LPAR).
All of the measurements were performed in completely expanded healthy leaves (four leaves per plant) randomly selected from the middle layer of the cacao canopy; four plants per AFs were assessed.
Photosynthetic light- and photosynthetic CO2-response curves in cacao under different AFs
Measurements were made using an infrared gas analyzer CIRAS-3 Portable Photosynthesis System (PP Systems Inc., Amesbury, MA, USA). The environmental conditions in the leaf cuvette consisted of a vapor pressure deficit (VPD) ranging from 1.0 to 1.5 kPa and a temperature of 25°C. Measurements were taken between 08:00 and 11:00 h (solar time), a partial concentration of CO2 of 400 ppm, and under artificial PAR provided by the LED light source of the cuvette.
The photosynthetic response curves to PAR intensity (henceforth, A/PAR) were generated by increasing PAR in 10 steps from 0 to 2,000 μmol m-2 s-1, a process that was carried out in a similar manner for the target cacao plants in each AF. Initially, the environmental conditions to which the leaves were exposed in the chamber were a VPD between 1.0 and 1.5 kPa, a leaf temperature of 25°C, and a partial concentration of CO2 of 50 ppm for 5 min to allow the stoma to open; subsequently A/PAR curves were obtained at a partial concentration of CO2 of 400 ppm. In order to determine the photosynthetic limitations of cacao that result from the microclimatic conditions in which it grows in each AF, the above data was used to determine different parameters derived from the A/PAR curves, such as light-saturated A (Amax), light compensation point (LCP), dark respiration rates (Rd), light saturation point (LSP), and apparent quantum efficiency (ΦPAR) determined from the slope of the initial linear portion of the A/PAR curve .
Photosynthetic response curves to CO2 concentration (henceforth, A/Ci) were determined at a PAR of 500 μmol m-2 s-1 (based on the A/PAR curves), at 25°C and ambient O2 concentration following the recommendations of Long and Bernacchi . Measurements were initiated at a partial concentration of CO2 of 400 ppm, which was gradually decreased to 50 ppm and subsequently increased in 15 steps up to 1,600 ppm of partial concentration of CO2 . Leakage errors were corrected by measuring the CO2 response curves in dead leaves following the recommendations of Flexas et al. . Different parameters derived from each A/Ci curve were determined including maximum rate of RuBisCO carboxylation (Vcmax), maximum rate of electron transport driving regeneration of RuBP (Jmax), and leaf respiration under light conditions (RD).
Chl a fluorescence parameters under the different AFs
Measurements were performed on the same leaves used to measure gas exchanges. This study used the chlorophyll fluorescence module (CFM-3) adapted for the infrared gas analyzer CIRAS-3 (PP Systems Inc., Amesbury, MA, USA). The CFM-3 provides chlorophyll fluorescence measurements using the pulse-amplitude modulation (PAM) technique. The maximum quantum yield of photosystem (PS) II (Fv/Fm) was determined at predawn (4:00 h; leaves in complete darkness) and by exposing the leaves to a saturating pulse of light (6,000 μmol m-2 s-1; 1s). After turning on a pulse of light, the leaf was exposed to a series of pulses of saturated light in order to obtain the maximum fluorescence yield in a light-adapted state (Fm′).
The actual PSII quantum efficiency (ΦPSII), which measures the proportion of light absorbed by the PSII light-harvesting antenna that is used in the photochemical process , was determined in the light-adapted state as follows: (1) where Fs is the fluorescence that was measured immediately before the application of pulses of light.
The apparent electron transport rate (ETR), which and is an indicator of overall photosynthetic capacity in vivo, was determined as follows : (2)
The fraction of PSII centers which are in the open state according to the Lake model of the PSII photosynthetic unit (qL) was obtained as follows : (3) where F0′ parameter is measured after an introduction of far-red illumination on light-adapted leaves (when all of the PSII reaction centers and electron acceptors are once again oxidized using a far-red light illumination).
Moreover, the non-photochemical quenching of Chl a fluorescence (NPQ), which provides an indication of thermal energy dissipation through the PSII light-harvesting antenna (or heat dissipation of Chl excitation energy), was estimated as follows : (4)
Specific leaf area, total chlorophyll, and nitrogen
Specific leaf area (SLA) was determined using six leaf discs (3.14 cm2) exclude of the mid-vein for each leaf that had been previously used to measure gas exchange and Chl a fluorescence (n = 96 per AFs or 6 x 4 x 4 –six discs per leaf x four leaves per plant x four plants per AFs). The discs were dried to constant mass at 70°C, and the SLA was determined as the ratio between leaf disc area and its respective dry mass .
We followed the protocol described by Lichtenthaler  to determine leaf contents of total Chl (Chlt), Chl a and Chl b, and carotenoids for each leaf taking other six discs (3.14 cm2) from the same leaves that had been previously used to measure SLA. The Kjeldahl method  was used to determine total nitrogen.
The Michaelis-Menten hyperbolic constant was used to adjust the A/PAR curves; the parameters Amax, LSP, LCP, Rd, and ΦPAR were calculated following the equations described in Lobo et al. . The model created by Farquhar et al.  (the ‘FvCB model’) was used to evaluate the A/Ci curve and estimate Vcmax, Jmax, and RD using the plantecophys package in R . A generalized linear model (GLM) was adjusted for the different parameters derived from the A/PAR and A/Ci curves for each cacao plant in each AFs (fixed factor). The plant and leaf were included as random factors (n = 16). Likewise, a GLM was made for SLA, photosynthetic pigments, and nitrogen content, including the AF as the fixed factor. The plant, leaf and discs were included as random factors. The assumptions of normality and of homogeneity of variance were evaluated using an exploratory residual analysis. Differences between mean cacao plant responses in the AFs (fixed factor) were analyzed with the Fisher’s LSD post-hoc test at a significance of α = 0.05. Analyses were performed using the lme function in the nlme package  in R language software, version 3.4.4 , and using the interface in InfoStat .
The different mean levels of incident PAR in the three agroforestry systems had an effect on the photosynthetic response of cacao, its functional traits (e.g., SLA), and the chlorophyll and N content in leaves (Table 1).
Parameters derived from photosynthetic light (A/PAR) and CO2 (A/Ci) response curves, specific leaf area, and pigments under different cacao agroforestry systems (AFs): cacaotal with Musaceae plantation and High mean daily incident radiation (HPAR), low diversity of clustered vegetation and Medium mean daily incident radiation (MPAR), and diversified multistrata vegetation and Low mean daily incident radiation (LPAR). The results include means ± SE (n = 4).
The cacao plants exhibited the greatest photosynthetic efficiency (Amax) in the AF with the highest amount of mean daily PAR (HPAR); cacao Amax in HPAR was double the value recorded in LPAR and higher than that recorded in MPAR (Fig 2, Table 1). On the other hand, the leaves of the cacao plants in the system with the lowest amount of PAR (LPAR) exhibited the highest values ΦPAR across AFs, whereas both LCP and LSP values were similar across AFs. The parameters derived from the A/Ci curves show that Vcmax reached its maximum in the HPAR, whereas the maximum rate of electron transport driving regeneration of RuBP (Jmax) was similar among cacao plants growing in HPAR and LPAR, but different from those growing in MPAR (Fig 3. Table 1).
A is the net carbon assimilation rate. The agroforestry systems for cacao were: cacaotal with Musaceae plantation and high mean daily PAR incident radiation (700 μmol m-2 s-1; HPAR), low diversity of clustered vegetation and medium mean daily PAR (400 μmol m-2 s-1; MPAR), and diversified multistrata vegetation and low mean daily PAR (300 μmol m-2 s-1; LPAR).
A is the net carbon assimilation rate, Ci is the intercellular CO2 concentration. Symbols represent the measured values, and the black line represents the fitted FvCB model of photosynthesis. Colored lines indicate the two photosynthesis rates in the FvCB model. Ac and Aj are the gross photosynthesis rates when RuBisCO activity and RuBP regeneration are limiting, respectively. a. cacaotal with Musaceae plantation and high mean daily PAR incident radiation (700 μmol m-2 s-1; HPAR), b. low diversity of clustered vegetation and medium mean daily PAR (400 μmol m-2 s-1; MPAR), c. diversified multistrata vegetation and low mean daily PAR (300 μmol m-2 s-1; LPAR).
The Fv/Fm mean values were similar for all AFs (0.81±0.01), suggesting that cacao plants in our study did not exhibit photosynthesis photoinhibition , whereas other Chl fluorescence parameters differed significantly between AFs (Fig 4). The electron transport rate (ETR) was higher in cacao plants cultivated in HPAR (8.5% and 18% higher compared to MPAR and LPAR, respectively, at a radiation of 1,500 μmol m-2 s-1) and similar in the MPAR and LPAR systems (Fig 4a). The PSII quantum efficiency (ΦPSII; Fig 4b) and the photochemical quenching coefficient (qL; Fig 4d) gradually decreased as the intensity of radiation increased, with the minimum values for the cacao plants recorded in the LPAR system. In the plants grown in the HPAR, the non-photochemical quenching (NPQ; Fig 4c) exhibited tendencies similar to those exhibited by the ETR results; however, more energy was dissipated in the form of heat—between 750 and 2,000 μmol m-2 s-1 of PAR.
(a) electron transport rate (ETR); (b) actual PSII quantum yield (ΦPSII); (c) non-photochemical quenching (NPQ) coefficient; (d) photochemical quenching (qL) coefficient. Each agroforestry system is depicted by one curve (continuous line) in conjunction with confidence levels (shaded). Legend of the agroforestry systems is the same as in Figs 1 and 2.
The SLA of cacao plants was inversely proportional to the mean incident PAR level across the different AFs, with SLA being 24.4% smaller in HPAR than in LPAR (Table 1). Radiation levels in the AFs had an effect on the content of photosynthetic pigments in cacao leaves. Generally, the leaves from LPAR exhibited higher leaf contents of Chlt (34.8%), Chl/Car ratio (53.2%), and N (45.5%) as compared to their counterparts from HPAR; the values recorded for MPAR were distinct, but fell in between those of the other two systems (Table 1). As for the leaf contents of Car and the Chl/N ratio, they were 34.4% and 19.6% higher, respectively, in HPAR than in LPAR and MPAR cacao plants. Lastly, the Chl a/b ratio was higher in MPAR cacao plants than in the other AFs, with plants in LPAR exhibiting the lowest ratios (Table 1).
In contrast to the assumption that cacao plants grow better under shade conditions, we showed that it exhibits optimal acclimatization to conditions of relatively high solar radiation. The optimal physiological performance of plants from HPAR is likely to have been associated with the high cloud cover that is typical of the Colombian Amazon, a region that displays and annual average of only 3–4 daily radiation hours . Irrespective, we showed compelling evidence demonstrating the ability of cacao plants to acclimate to HPAR conditions: i. overall, these plants displayed a decrease in Chlt as well as an increase in Chl a/b ratio; in parallel HPAR plants also showed a decrease in Chl/Car ratio and higher NPQ values, which favor the dissipation of excess energy in the form of heat [34–37]; ii. despite the high solar radiation the HPAR plants did not show chronic photosynthesis photoinhibition, as denoted by the values of Fv/Fm ratio above 0.80 ; iii. these plants displayed the highest Amax (even on a per leaf mass basis) and Vcmax as compared with the plants from the other AFs, suggesting higher RuBisCO activity . Collectively, these acclimative responses to high solar radiation, in addition to improving the photosynthetic performance, should also contribute to better growth and crop yields in HPAR than in MPAR or LPAR conditions.
In contrast to be HPAR plants, their LPAR counterparts showed high contents of photosynthetic pigments coupled with an increase in leaf N content and Chl/N ratio; these responses were probably related to adjustments in light harvesting ability. Both LPAR and MPAR plant showed a decrease in Rd and an increase in ΦPSII, which have also been reported for other species subjected to low irradiance and are considered as an adaptation strategy for survival under shade conditions. The increase in SLA is an additional strategy to cope with low PAR. In this regard, the greater SLA exhibited by cacao plants grown in the LPAR might be considered as a mechanism to maximize photon capture efficiency, which may improve photosynthetic capacity  and carbon gain , in addition to concentrating resources invested in the construction of photosynthetic tissues . This adaptation in plants subjected to low light intensities seems to be related to a decrease in leaf thickness and increase in leaf area for a more efficient absorption and optimization of photon capture . However, as stated, these morphological (e.g., high SLA) and biochemical (increased leaf N content) adjustments of the highly shaded plants in LPAR were not capable of improving its photosynthetic performance, as has also been observed in other studies conducted with different plant species under shady conditions .
Overall, many studies [5, 8, 11, 16, 35, 38] have demonstrated that cacao plants exhibit an optimal physiological behavior under conditions of low radiation, specifically at mean PAR levels of 400 μmol m-2 s-1, contrasting with what was found in our study. Results show that cacao plantations in HPAR with mean radiation levels of ca. 700 μmol m-2 s-1 increased their Amax compared to plants from MPAR or LPAR. Our results are in agreement with Jaimez et al.  who showed similar photosynthetic performance in cacao plants; other studies conducted in regions with high humidity and cloudy conditions point to similar results, such as Bently et al.  in the Amazonian Ecuador and de Araujo et al.  in the Atlantic coast of Brazil. Studies carried out under greenhouse conditions also demonstrate the adaptation of cacao to high radiation levels (1,200 μmol m-2 s-1) with A values of 5.7 μmol m-2 s-1 . Taken together, these facts suggest that, under the conditions of the Colombian Amazon, the cacao plant has the capacity to acclimate to high light levels.
This study shows that cacao plants growing under HPAR exhibit improved carbon assimilation performance, demonstrating photosynthetic acclimatization to patterns of high solar radiation that resulted in higher Amax and Vcmax than in plants from the MPAR or LPAR systems. Even though the cacao plant is considered a demanding shade species, this study suggests that under the conditions of the Colombian Amazon, cacao plants can be even grown in monoculture instead of in AFs. Nonetheless, the high cloud cover conditions that prevail in the Colombian Amazon can differ from the other regions worldwide where cacao is grown; this could explain some discrepancies found in the literature as regarded to the optimal light conditions for the successful growth and production of cacao plants. Our results also suggests the need of case-by-case evaluation for the optimal conditions for cacao growth, taking into account the specific climatic conditions of a given geographic region. Given that cacao is one of the most important perennial crops in the world and its cultivation is a major economic resource in many developing regions, the provision of proper recommendations to the local producers is crucial for the sustainable implementation of agronomical practices resulting in improved crop yields.
S1 File. Leaf photosynthesis-CO2 response curves of cacao for HPAR.
S2 File. Leaf photosynthesis-CO2 response curves of cacao for MPAR.
S3 File. Leaf photosynthesis-CO2 response curves of cacao for LPAR.
S4 File. The photosynthetic response curves to PAR intensity for AFs.
We thank Rocio L. Labrador for her English edition and reviewers and the Academic Editor for their comments on early versions of this manuscript.
- 1. ICCO (International Cocoa Organization). Quarterly Bulletin of Cocoa Statistics, Vol. XL, No. 1, Cocoa year 2015/16. 2017.
- 2. Motamayor J, Lachenaud P, da Silva e Mota J, Loor R, Kuhn D, Brown J, et al. Geographic and genetic population differentiation of the Amazonian chocolate tree (Theobroma cacao L). PLoS One. 2008;3(10):e3311. pmid:18827930
- 3. Baker R, Cope F, Holliday P, Bartley B, Taylor J. The Anglo-Colombian cacao collecting expedition. Repr Arch Cocoa Res Vol 1, ed Toxopeus H (Vienna, VA Am Cocoa Res Institute) 1952.
- 4. Carr M, Lockwood G. The water relations and irrigation requirements of cocoa (Theobroma cacao L.): a review. Exp Agric. 2011;47(4):653–76.
- 5. Almeida A, Gomes F, Araujo R, Santos R, Valle R. Leaf gas exchange in species of the Theobroma genus. Photosynthetica. 2014;52(1):16–21.
- 6. Daymond A, Tricker P, Hadley P. Genotypic variation in photosynthesis in cacao is correlated with stomatal conductance and leaf nitrogen. Biol Plant. 2011;55(1):99–104.
- 7. Avila-Lovera E, Coronel I, Jaimez R, Urich R, Pereyra G, Araque O, et al. Ecophysiological traits of adult trees of criollo cocoa cultivars (Theobroma cacao L.) from a germplasm bank in Venezuela. Exp Agric. 2015;52(1):137–53.
- 8. Tezara W, Urich R, Jaimez R, Coronel I, Araque O, Azocar C, et al. Does Criollo cocoa have the same ecophysiological characteristics than Forastero? Bot Sci. 2016;94(3):563–74.
- 9. Mielke M, Almeida A, Gomes F. Photosynthetic traits of five neotropical rainforest tree species: interactions between light response curves and leaf-to-air vapour pressure deficit. Brazilian Arch Biol Technol. 2005;48(5):815–24.
- 10. Tezara W, Almeida A, Valencia E, Cortes J, Bolaños M. Actividad fotoquímica de clones élites de cacao (Theobroma cacao L.) Ecuatoriano en el norte de la provincia Esmeraldas. Investig y Saberes. 2015;4(3):563–74.
- 11. Acheampong K, Hadley P, Daymond A. Photosynthetic activity and early growth of four cacao genotypes as influenced by different shade regimes under west african dry and wet season conditions. Exp Agric. 2013;49(1):31–42.
- 12. IDEAM. Instituto de Hidrología Meteorología y Estudios Ambientales. Atlas Climatológico de Colombia. IDEAM, Bogotá, D.C. 2018. 219 p.
- 13. Galyuon I, McDavid C, Lopez F, Spence J. Effect of irradiance level on cocoa (Theobroma cacao L.). I. Growth and leaf adaptations. Trop Agric. 1996;73:23–8.
- 14. Galyuon I, McDavid C, Lopez F, Spence J. Effect of irradiance level on cocoa (Theobroma cacao L.). II. Gas exchange and chlorophyll fluorescence. Trop Agric. 1996;73:29–33.
- 15. DaMatta F, Ramalho J. Impacts of drought and temperature stress on coffee physiology and production: a review. Braz J Plant Physiol. 2006;18:55–81.
- 16. Araque O, Jaimez R, Tezara W, Coronel I, Urich R, Espinoza W. Comparative photosynthesis, water relations, growth and survival rates in juvenile criollo cacao cultivars (Theobroma cacao L.) during dry and wet seasons. Exp Agric. 2012;48(4):513–522.
- 17. Suárez JC, Bieng MA, Melgarejo LM, Di Rienzo JA, Casanoves F. First typology of cacao (Theobroma cacao L.) systems in Colombian Amazonia, based on tree species richness, canopy structure and light availability. PLoS One. 2018;13(2):e0191003. pmid:29401499
- 18. Balasimha D, Apshara S, Jose C. Genotypic variations in chlorophyll fluorescence and stomatal conductance of cocoa in relation to drought tolerance. J Plant Crop. 2013;41(1):40–5.
- 19. Daymond A, Hadley P. The effects of temperature and light integral on early vegetative growth and chlorophyll fluorescence of four contrasting genotypes of cacao (Theobroma cacao). Ann Appl Biol. 2004;145(3):257–62.
- 20. Bauerle W, Wang G, Bowden J, Hong C. An analysis of ecophysiological responses to drought in American chestnut. Ann For Sci. 2006;63(8):833–42.
- 21. Long S, Bernacchi C. Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error. J Exp Bot. 2003;54(392):2393–401. pmid:14512377
- 22. Martins S, Detmann K, Reis J, Pereira L, Sanglard L, Rogalski M, et al. Photosynthetic induction and activity of enzymes related to carbon metabolism: insights into the varying net photosynthesis rates of coffee sun and shade. Theor Exp Plant Physiol. 2013;25(1):62–9.
- 23. Flexas J, Díaz-Espejo A, Berry JA, Cifre J, Galmés J, Kaldenhoff R, et al. Analysis of leakage in IRGA’s leaf chambers of open gas exchange systems: Quantification and its effects in photosynthesis parameterization. J Exp Bot. 2007;58(6):1533–43. pmid:17339650
- 24. Genty B, Briantais J, Baker NR. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta. 1989;990(1):87–92.
- 25. Brooks MD, Niyogi KK. Use of a Pulse-Amplitude Modulated Chlorophyll Fluorometer to Study the Efficiency of Photosynthesis in Arabidopsis Plants. In: Jarvis R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology (Methods and Protocols), vol 775. Humana Press, Totowa, NJ. 2011. p. 299–310.
- 26. Cornelissen JHC, Lavorel S, Garnier E, Díaz S, Buchmann N, Gurvich DE, et al. A handbook of protocols for standardised and easy measurement of plant functional traits worldwide. Vol. 51, Aust J Bot. 2003. p. 335–80.
- 27. Lichtenthaler HK. Chlorophyls and carotenoids: Pigments of photosynthetic biomembranes. Meth Enzimol. 1987;148(34):350–82.
- 28. Lobo F de A, de Barros MP, Dalmagro HJ, Dalmolin ÂC, Pereira WE, de Souza ÉC, et al. Fitting net photosynthetic light-response curves with Microsoft Excel—a critical look at the models. Photosynthetica. 2013;51(3):445–56.
- 29. Farquhar GD, von Caemmerer S, Berry JA. A biochemical-model of photosynthetic CO2 assimilation in eaves of C-3 species. Planta. 1980;149(1):78–90.
- 30. Duursma RA. Plantecophys—An R package for analysing and modelling leaf gas exchange data. Struik PC, editor. PLoS One. 2015 Nov 18;10(11):e0143346. pmid:26581080
- 31. Pinheiro J, Bates D, Debroy S, Sarkar D. Linear and Nonlinear Mixed Effects Models. R Packag version. 2012;3(1–104):3.1–74.
- 32. R Development Core Team. R: A language and environment for statistical computing. R. Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/. Citeseer; 2018.
- 33. Di Rienzo J, Balzarini M, Gonzalez L, Casanoves F, Tablada M, Robledo CW. InfoStat versión 2018. Grupo infostat, FCA, Universidad Nacional de Córdoba, Argentina. URL http://www.infostat.com.ar. 2018.
- 34. Fu Y, Li H, Yu J, Liu H, Cao Z, Manukovsky NS, et al. Interaction effects of light intensity and nitrogen concentration on growth, photosynthetic characteristics and quality of lettuce (Lactuca sativa L. Var. youmaicai). Sci Hortic (Amsterdam). 2017;214:51–7.
- 35. Pompelli MF, Martins SC, Antunes WC, Chaves ARM, DaMatta FM. Photosynthesis and photoprotection in coffee leaves is affected by nitrogen and light availabilities in winter conditions. J Plant Physiol. 2010;167(13):1052–60. pmid:20381192
- 36. de Araújo RP, de Almeida AAF, Barroso JP, de Oliveira RA, Gomes FP, Ahnert D, et al. Molecular and morphophysiological responses cocoa leaves with different concentrations of anthocyanin to variations in light levels. Sci Hortic (Amsterdam). 2017;224:188–97.
- 37. Armbruster U, Correa Galvis V, Kunz HH, Strand DD. The regulation of the chloroplast proton motive force plays a key role for photosynthesis in fluctuating light. Vol. 37, Current Opinion in Plant Biology. Elsevier Current Trends; 2017. p. 56–62.
- 38. Vialet-Chabrand S, Matthews JSA, Simkin AJ, Raines CA, Lawson T. Importance of Fluctuations in Light on Plant Photosynthetic Acclimation. Plant Physiol. 2017;173(4):2163–79. pmid:28184008
- 39. da Silva Branco MC, de Almeida AAF, Dalmolin ÂC, Ahnert D, Baligar VC. Influence of low light intensity and soil flooding on cacao physiology. Sci Hortic (Amsterdam). 2017;217:243–57.
- 40. Russo SE, Kitajima K. The Ecophysiology of Leaf Lifespan in Tropical Forests: Adaptive and Plastic Responses to Environmental Heterogeneity. In: Tropical Tree Physiology. Springer International Publishing; 2016. p. 357–83.
- 41. Chaves ARM, Ten-Caten A, Pinheiro HA, Ribeiro A, Damatta FM. Seasonal changes in photoprotective mechanisms of leaves from shaded and unshaded field-grown coffee (Coffea arabica L.) trees. Trees—Struct Funct. 2008;22(3):351–61.
- 42. Chmura DJ, Modrzyński J, Chmielarz P, Tjoelker MG. Plasticity in seedling morphology, biomass allocation and physiology among ten temperate tree species in response to shade is related to shade tolerance and not leaf habit. Adams W, editor. Plant Biol. 2017 Mar;19(2):172–82. pmid:27981788
- 43. Liu Y, Dawson W, Prati D, Haeuser E, Feng Y, Van Kleunen M. Does greater specific leaf area plasticity help plants to maintain a high performance when shaded? Ann Bot. 2016;118(7):1329–36. pmid:27594648
- 44. Jaimez RE, Amores Puyutaxi F, Vasco A, Loor RG, Tarqui O, Quijano G, et al. Photosynthetic response to low and high light of cacao growing without shade in an area of low evaporative demand. Acta Biol Colomb. 2018;23(1):95–103.
- 45. Bentley J. W., Boa E., & Stonehouse J. Neighbor trees: shade, intercropping, and cacao in Ecuador. Human Ecology. 2004;32(2):241–270.