Morpho-physiological performance of Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker plants under different light conditions

ABSTRACT

(Morpho-physiological performance of Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker plants under different light conditions). In tropical and subtropical zones, lianas play important roles in the process of ecological succession. This study aims to evaluate the photosynthetic and morpho-physiological performance between two lianas species from Mikania genus in response to different levels of radiation: full sun (I0), 25% (I25), 50% (I50), and 75% (I75) retention of solar radiation flux. Plants grown under I75 showed a reduced net photosynthetic rate (A). We observed dynamic photoinhibition at I0 during hours of high irradiation and temperature. The highest and lowest leaf chlorophyll content occurred at I75 and I0, respectively, while carotenoids/total chlorophyll and leaf thickness increased under I0. Total dry mass was higher in plants grown at I0 and I25. However, A values and biomass production of Mikania laevigata were higher at I25, while for Mikania glomerata greater biomass accumulation was observed between I0-I50. Therefore, we concluded that M. laevigata and M. glomerata have different morpho-physiological performances under same the radiation conditions.

Keywords:
chlorophyll a fluorescence; gas exchange; irradiation interference; lianas; photosynthesis

RESUMO

(Desempenho morfo-fisiológico de Mikania glomerata Spreng. e Mikania laevigata Sch. Bip ex Baker sob diferentes condições de luminosidade). Em regiões tropical e subtropical, lianas desempenham um papel importante no processo de sucessão ecológica. O objetivo deste estudo foi avaliar as respostas fotossintéticas e morfo-fisiológicas entre duas espécies de Mikania em diferentes níveis de radiação: sol pleno (I0), 25% (I25), 50% (I50) e 75% (I75) de retenção do fluxo da radiação solar. Plantas crescidas sob I75 mostraram reduzida taxa fotossintética (A). Nós observamos fotoinibição dinâmica em plantas crescidas sob I0 durante as horas de alta irradiação e temperatura. O maior e menor conteúdo de clorofila ocorreu em plantas sob I75 e I0, respectivamente; enquanto carotenoides/clorofila total e espessuras da epiderme e mesofilo aumentaram sob I0. A massa seca total foi maior em plantas crescidas sob I0 e I25. No entanto, os valores de A e a produção de biomassa de M. laevigata foram maiores sob I25; enquanto para M. glomerata, maior acúmulo de biomassa foi observado entre I0-I50. Portanto, nós concluímos que M. laevigata e M. glomerata apresentaram diferentes respostas morfo-fisiológicas sob mesma condição de radiação.

Palavras-chave:
fluorescência da clorofila a; fotossíntese; interferência da irradiação; lianas; trocas gasosas

Introduction

Lianas are life forms frequently found in tropical and subtropical forests (Gentry & Dodson 1987) that explore all forest layers through different mechanisms of ascension, growth patterns, and physiological strategies (Schnitzer & Bongers 2002, Gerwing 2004, Kazda et al. 2009). Lianas stems are relatively thin and depends on external support to access the sunlight. Therefore, lianas species often allocate less carbon in stem growth, and more carbon in photosynthetic and vascular tissues (Putz 1984, Schnitzer 2005), leading to an advantage over trees due to higher growth rates (Zhu & Cao 2009). In addition, the ability to grow both laterally and vertically allows lianas to easily invade the canopy, extending long branches and reaching adequate light conditions (Schnitzer & Bongers 2002, Toledo et al. 2003). Since radiation requirements of lianas are high, those species are often classified as gap-dependent pioneer species, presenting a similar distribution pattern of pioneer tree species (Putz 1984, Schnitzer & Bonger 2002). However, some lianas are also able to germinate and grow in the understory (Nabe-Nielsen 2002, Sanches & Válio 2002, Schnitzer et al. 2012), suggesting some level of shade tolerance (Gerwing 2004).

In the last decades, lianas abundance and productivity increased in tropical forests especially due to high rates of deforestation and human-induced climate change (Granados & Korner 2002, Phillips et al. 2002, Wright et al. 2004, Zhu et al. 2004, Schnitzer & Bongers 2011). In addition, evidences indicated that in mature and undisturbed forests the increased density of lianas may be consequence of changes in precipitation patterns (Schnitzer & Bongers 2011). Another important factor that influences lianas growth and distribution is light availability. For example, it is recognized that increased solar radiation improves lianas seedlings growth rates (Kurzel et al. 2006, Schnitzer & Bongers 2011). Furthermore, lianas may experience different amounts of radiation and spectral quality during growth. Therefore, it is expected a high phenotypic plasticity of lianas regarding photosynthetic and gas exchange adjustments in response to different light conditions (Bazzaz & Carlson 1982, Ribeiro et al. 2005). Stomatal opening level determines the trade-off between CO₂ absorption and water loss by transpiration (Caemmerer & Baker 2007). The adaptative success under different light conditions depends on adjustments of leaf morphology, anatomy, and photosynthetic apparatus. Therefore, adjustments ensure greater efficiency in the conversion of radiant energy into carbohydrates to sustain plant growth (Dias-Filho 1997, Campos & Uchida 2002, Gratani 2014).

Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker are lianas species both native from Atlantic Forest in Brazil (Gasparetto et al. 2010). Mikania species belong to the Asteraceae family and are popularly known as “Guaco” and they both can benefit from Atlantic forest fragmentation. Within biodiversity hotspots around the world, the Atlantic forest is considered the most vulnerable ecosystem to deforestation and climate-change (Béllard et al. 2014). With a wide range of forest physiognomies (Myers et al. 2000, Ricketts et al. 2005, Metzger 2009), currently, only 28% of its original area remains (Rezende et al. 2018). Both species are similar regarding its morphology and are often indiscriminately used in traditional medicine to treat colds, flu, asthma, and, bronchitis because of the bronchodilator and expectorant properties (Moura et al. 2002, Graça et al. 2007, Bolina et al. 2009, Gaspareto et al. 2010). Recent studies demonstrated that M. glomerata and M. laevigata have a different chemical composition and therapeutic properties (Melo & Sawaya 2015, Almeida et al. 2016, Costa et al. 2017). However, no reports of photosynthetic proprieties and gas exchange behavior under different light conditions between both species are found in the literature.

Therefore, our objective was to evaluate the morpho-physiological performance of two Mikania species (Mikania glomerata and Mikania laevigata) under four different levels of retention of solar radiation flux: full sun (I0), and 25 (I25), 50 (I50), and 75% (I75). Our main hypothesis is that both Mikania species will be benefited from high radiation levels, resulting in enhanced photosynthesis and biomass production.

Materials and methods

Plant material and growth conditions - Experiments were carried out at the University of São Paulo at Ribeirão Preto campus (21° 10 ‘08.4’’ 102 S and 47° 51’ 50.6’’ W), São Paulo, Brazil, which climate is tropical wet and dry (Aw) according to the Köppen-Geiger classification. For further details of climatic conditions during the experiment (Supplementary material Table S1). We used the species Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker. Mikania plantlets from each species were prepared cuttings from the middle of the branches of different parental plants of approximately 1 cm in diameter, 12 cm in length, a node at the top of the stake, and a pair of leaves (Lima et al. 2003). The plantlets were planted in 3-kg plastic bags containing a mixture of manure and soil (soil type redlatosol, 1:1) under greenhouse conditions. After rooting (approximately 60 days), the plants were transferred into 20 L pots containing soil and submitted to treatments for 150 days. The soil was fertilized with 1 g NPK (4-14-8) fertilizer per kg of soil. Plants were subjected to four light conditions: full sun condition (I0), and 25% (I25), 50% (I50), and 75% (I75) retention of solar radiation flux (Supplementary material Figure S1). To achieve the planned levels of solar radiation, special greenhouses were constructed with artificial shading of varying degrees of retention of solar radiation flux. During the experiment, pots were irrigated daily and were maintained at soil field capacity, using a sensor ML2× Theta Probe (Delta-T Devices, Cambridge, UK).

Gas exchange parameters, chlorophyll fluorescence, and photosynthetic pigment content were evaluated after 60, 90, 120, and 150 days after the treatments started (DAT). Leaf anatomy and biomass production were evaluated at 90 and 150 DAT. Three leaf samples per plant from upper, middle and lower canopy regions were collected.

Gas exchange measurements - Net photosynthesis rate (A, µmol m^-2 s^-1), transpiration rate (E, mmol m^-2 s^-1), and stomatal conductance (g_s, mol m^-2 s^-1) were evaluated daily from 8:00 to 11:00 hours in fully expanded leaves using an infrared gas analyzer model LCpro⁺ (ADC BioScientific, Ltd., UK). The measurements were made under ambient conditions of radiation, [CO₂], and air temperature. The intrinsic water-use efficiency (_iWUE; µmol mol^-1) was estimated from gas exchange (A/g_s) data.

Chlorophyll fluorescence - The maximum quantum yield of primary photochemistry (Fv/Fm) was measured in three fully expanded leaves using a portable fluorometer model OS-3P (ADC BioScientific, UK). Leaves were dark-adapted for 30 minutes and used to measure the dark fluorescence yield (Fo), maximum fluorescence yield (Fm), and variable fluorescence (Fv). Then, Fv/Fm ratio was calculated. We performed four Fv/Fm diurnal courses from 6:00 hours to 18:00 hours each two hours at 60, 90, 120, and 150 DAT. At sampling days, we monitored the relative humidity and ambient temperature using a hygro-thermometer, and the photosynthetic photon flux density (PPFD) using a quantum sensor connected to an irradiation meter model LI-250A (LI-COR, USA) (Supplementary material Figure S1).

Photosynthetic pigment analysis - Photosynthetic pigments were extracted and quantified following the methodology of Hendry and Price (1993). Leaf discs (0.1g) were ground in 80% acetone, and the absorbance was measured at 480, 645, and 663 nm using a spectrophotometer model Genesys 5Spectronic. Based on the absorbance value, the concentration of total chlorophyll and carotenoids were calculated.

Leaf anatomy - Leaf fragments (1 cm²) were fixed in FAA 70% for 24 hours and dehydrated in ethanol series (Kraus & Arduin 1997). Then, samples were embedded in paraffin, cut in a microtome (8 μm), and stained with 1% toluidine blue. Samples were observed using a microscope QUIMIS (Q720 ED), photographed and the images were used to measure the leaf thickness, adaxial (AdE) and abaxial epidermis thickness (AbE), and palisade (PP) and spongy parenchyma (SP) thickness. Measurements were performed using the software AnatiQuanti 2.0 (Laboratory of Plant Anatomy/UFV). We found a hypodermic layer below the adaxial epidermis in both species, but since we did not found this tissue in all samples, it was not quantified.

For the epidermis analysis, lower epidermis (hypoestomatic leaves) were detached from mesophyll using the Jeffrey solution (10% chromic acid and 10% nitric acid, 1:1). Samples were stained with safranin for 30 seconds and mounted in glycerin 50% (Kraus & Arduin 1997). Samples were observed using a microscope QUIMIS (Q720 ED) and photographed. We counted the number of epidermal cells and stomata and calculated the stomatal density (SD) and stomatal index (SI) using the software AnatiQuanti 2.0 (Laboratory of Plant Anatomy/UFV).

Stomatal index (SI) was calculated according to the equation:

$$SI=\frac{SN}{SN+EC}\ast 100$$

where: SN = stomata number; EC = number of epidermal cells

Leaf area and biomass analysis - To measure leaf area (LA), we detached leaves from the whole plant and detached leaf discs of 1 cm² from basal, median, and apical regions of the leaves. Using the disc area, disc dry weight, and total leaf dry weight, we estimated the mean leaf area. Specific leaf area (SLA) was estimated as the ratio of leaf area to leaf dry mass (dm² g^-1).

For biomass, five plants from each treatment were collected. Samples were separated in roots, stems, petiole, and leaves. Then, plant material was dried at oven (70 °C) until constant mass. Subsequently, the dry weight of each organ was determined.

Experimental design and statistical analysis - The effects of irradiance interference, species, and their interactions were evaluated using analysis of variance (two-way ANOVA) using the SYSTAT software package (SPSS Inc., Chicago, IL) (P < 0.05 was accepted as statistically significant). Significant effects were further analyzed using Tukey’s test. The ANOVA included two species (M. glomerata and M. laevigata) and four levels of sunlight retention of solar radiation flux treatment: 0% (I0), 25% (I25), 50% (I50), and 75% (I75).

Results

Gas exchange - The solar radiation level significantly affected stomatal conductance (g_s) and transpiration rate (E) regardless of species, except at 150 DAT (figure 1 a-d; ANOVA in Supplementary material Table S2). E and g_s values were lower in plants grew under higher retention of solar radiation flux (I75). At 150 DAT, g_s showed the lowest values when compared to previous samplings. When species were compared, g_s was higher in M. laevigata than in M. glomerata (Supplementary material Table S2).

Net photosynthetic rate (A) from both Mikania species was significantly affected by solar radiance levels and species. However, no interactions between factors were observed for A (figure 1 e-f). Compared with plants grown under high solar radiances, a lower A was observed in plants grown under the greatest shading (I75) in both species (figures 1 e-f). At 150 DAT and under I75, A showed the lowest values and was 6.7 and 4.5-fold lower than plants under I25, in Mikania glomerata and Mikania laevigata, respectively. On average, in M. glomerata and M. laevigata, the highest A (12.69 and 14.87 µmol m^-2 s^-1, respectively) was observed in plants grown under I25 compared with other treatments (figure 1 e-f). During the entire experiment, the lowest A and g_s values were observed at 150 DAT, period with low air relative humidity, highest vapor pressure deficit (VPD), and increased air temperature (Supplementary material Table S3).

Intrinsic water-use efficiency (_iWUE) of M. glomerata plants grown at I75 was higher than at I0 (figure 1 g). However, in M. laevigata plants grew under I75, _iWUE showed the lowest values when compared to other treatments (figure 1 h). Lower values of _iWUE were observed in M. laevigata plants when compared with M. glomerata plants (Supplementary material Table S2). This result might reflect the increased g_s observed in M. laevigata plants (figure 1 h).

Diurnal courses of chlorophyll a fluorescence - In both species, and under high shading conditions (I75), Fv/Fm values were higher compared with all other treatments (figure 2). We observed that in both species at 60 and 90 DAT only plants developed under I0 showed a decreased Fv/Fm ratio, with values below 0.75, indicating the occurrence of dynamic photoinhibition (figure 2 a-d). At 120 and 150 DAT, Fv/Fm reduction was observed under all treatments, except under I75 (figure 2 e-h). The recovery of this parameter was observed at 18:00 hours, except in M. glomerata plants grown under I0 and I25, and M. laevigata I0 (150 DAT), indicating a lower rate of recovery. In all treatments, the lowest Fv/Fm value was observed between 12:00 and 16:00 hours in plants grown under the full sun (figure 2).

Photosynthetic pigments - The effects of retention of solar radiation flux on the photosynthetic pigments were similar in both species. There were significant effects of solar radiances and species on total chlorophyll and carotenoids for both species (figure 3; Supplementary material Table S2). Pigments concentration increased under low light availability. Plants grew under I0 showed a reduction in chlorophyll and carotenoids content during the experiment (figure 3 a-d). For M. glomerata, 5.6 and 4.0-fold more chlorophyll and carotenoids were found for I75 than I0 treatments at the end of the experiment (150 DAT) (figure 3 a, c). In M. laevigata plants, this difference was between 4.9 and 3.6-fold. In this same sampling, both species under I25 showed an average increase of 3-fold more chlorophyll and 2.5-fold more carotenoids than plants under I0 (figure 3 b, d). In both species, higher values of carotenoids/chlorophyll ratio were observed in plants grown in I0 compared with plants grown under high levels of shading (I75) (figure 3 e, f).

Figure 1
Gas exchange parameters: Stomatal conductance (g_s, a-b), transpiration rate (E, c-d), net photosynthesis (A, e-f), and intrinsic water use efficiency (iWUE, g-h) in Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker, at 60, 90, 120 and 150 days after the treatment started (DAT), grown under sunlight irradiation interference treatments: 0% (I0), 25% (I25), 50% (I50) and 75% (I75). Data shown are the means (± SE) for measurements made on five plants per treatment.

Figure 2
Diurnal course of maximum quantum efficiency of PSII (Fv/Fm) in Mikania glomerata Spreng. at 60 (a), 90 (c), 120 (e), and 150 (g) days after the treatment started (DAT) and Mikania laevigata Sch. Bip ex Baker at 60 (b), 90 (d), 120 (f), and 150 DAT (h), grown under sunlight irradiation interference treatments: 0% (I0), 25% (I25), 50% (I50), and 75% (I75). Data shown are the means (± SE) for measurements made on five plants per treatment.

Figure 3
Photosynthetic pigments: Content of total chlorophyll (a-b), total carotenoids (c-d), and total carotenoids/chlorophyll ratio (e-f) in Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker, at 60, 90, 120, and 150 days after the treatment started (DAT), grown under sunlight irradiation interference treatments: 0% (I0), 25% (I25), 50% (I50), and 75% (I75). Data shown are the means (± SE) for measurements made on five plants per treatment.

Leaf anatomy - The effects of retention of solar radiation flux on leaf anatomy were similar in both species. The thickness of the adaxial epidermis (AdE) and mesophyll (palisade and spongy parenchyma) decreased while the retention of solar radiation flux increased (table 1). The AdE was approximately 1.2 and 1.3-fold higher in I0 compared with I75 for M. glomerata and M. laevigata, respectively. Under I0, I25, and I50 the AdE was on average 1.4 and 1.7-fold higher in M. laevigata than M. glomerata at 90 and 150 DAT, respectively. Under I75, M. laevigata also showed a higher AdE. However, the values were 1.2 and 1.4-fold (90 and 150 DAT, respectively) higher than those observed in M. glomerata. Under I0 palisade and spongy parenchyma (150 DAT), were 10 and 17% higher in M. laevigata (table 1).

Stomatal index (SI) and stomatal density (SD) both increased according to the increased levels of radiation (table 2). The highest SI and SD values were observed at 150 DAT for both species under I0 and I25. In M. glomerata grew under I0, SI and SD were 18.05% and 256 stomata/mm², respectively, while for M. laevigata developed under I0, SI and SD were 14.41% and 176 stomata/mm², respectively. Regardless of the solar radiances level, M. glomerata showed the highest values of SI and SD when compared to M. laevigata.

Morphology and biomass partitioning - There was a significant effect of the retention of solar radiation flux and species for leaf area (LA). M. glomerata showed higher LA than M. laevigata at 90 and 150 DAT (figure 4). At 90 DAT, LA of plants developed under I0 was 50% and 12% higher than plants grew under I75 for M. glomerata and M. laevigata, respectively (figure 4 a). At 150 DAT, this difference was between 23% and 27%. In this same sampling, LA of M. glomerata developed under I0, I25, and I50 were 1.7-fold higher when compared to M. laevigata plants (figure 4 b). The SLA decreased with increased levels of solar radiances (figure 4 c, d). In both species (150 DAT) developed under I75, SLA was on average 1.4-fold higher when compared to plants grown under I0, I25, and I50 (figure 4 d).

The highest production of aboveground biomass was observed at 90 DAT in M. glomerata under I50 (45.37 g DW), and for M. laevigata under I0 (28.19 g DW) (figure 5 a). At 150 DAT, shoot biomass in M. glomerata was higher under I0, followed by plants grew under I25 and I50 (figure 5 b). However, in M. laevigata plants, the highest aboveground dry mass was observed in plants developed under I25 followed by I0. At 90 and 150 DAT, we observed that under I0, root dry mass was higher compared to other treatments and independent of species (figure 5). Both species at 90 DAT showed higher leaf biomass production when compared to other part plants, however, at 150 DAT, the stem biomass was higher than leaf dry mass (figure 5). At the end of the experiment (150 DAT), increased production of biomass was observed under I25 in M. laevigata compared with other solar radiances treatments. In M. laevigata, leaf, stem, and root biomass of plants grown under I75 were 38, 48, and 70%, respectively, lower when compared with plants grown under I25. Compared with full sun treatments (I0), root biomass showed a 54% and 69% reduction under I50 and I75 treatments, respectively (figure 5).

In M. glomerata plants, leaf and stem biomass were on average 40 and 48% higher under I0, I25, and I50 than under I75. Root biomass was 56, 40, and 70% higher under I0 when compared to plants grown under I25, I50, and I75, respectively. Moreover, we observed that M. glomerata plants grown under I0, I25, and I50 had greater leaves and stems biomass than those observed in M. laevigata plants (figure 5).

Discussion

In this study, we unraveled the main morpho-physiological characteristics of two tropical lianas species in response to light availability. Our main hypothesis was not corroborated, since Mikania glomerata showed a better growth performance under I0, I25, and I50, while Mikania laevigata showed improved performance under I25. Plants grown under I0 and I25 showed greater g_s and E. The increased air temperature and VPD observed at 150 DAT presumably caused the reduction in g_s, E, and A in both Mikania species. Stomatal closure under high light conditions combined with high temperature, and low relative humidity it is a mechanism that decreases the water loss rate to the environment, but it also decreases the CO₂ influx into the leaves (Hsie et al. 2015). Stomatal regulation is an advantageous strategy, especially during the dry season. Under field conditions, improved _iWUE due to the reduction of g_s and constant A values is the most effective mechanism for the continued growth of plants without experiencing dramatic water loss (Hanba et al. 2002).

Our data indicated that M. glomerata and M. laevigata plants grown under conditions of retention of solar radiation flux of up to 50% had higher A than plants grown under I75. Lower A under I75 is presumably the result of a combination of many factors such as thinner leaves, low stomatal density, decreased g_s, and low light intensity. These factors may result in lower intercellular CO₂ concentration (C_i), Rubisco activity, and electron transporters (Lambers et al. 1998). Notably, plants grown under I25 exhibited higher A values than plants grown under full sunlight (I0). This effect was observed in both species, suggesting that the irradiation growth condition was too high at I0, which potentially induced dynamic photoinhibition. Fv/Fm is a parameter that indicates photoinhibition level, and smaller values than 0.75 indicate photoinhibition with a concomitant reduction of the maximum quantum efficiency of PSII (Bolhàr-Nordenkampf & Öquist 1993). Under full sunlight conditions, the reduced Fv/Fm at noon and subsequent recovery at the end of the day is evidence of dynamic reversible photoinhibition (Bolhàr-Nordenkampf & Öquist 1993). The dynamic photoinhibition (inactivation of PSII) is an efficient defense mechanism because the increase in non-photochemical dissipation when the influx of CO₂ is reduced (due to stomatal closure), prevents the formation of reactive oxygen species (ROS) and photooxidative damage (Choudhury & Behera 2001).

Differences in the level of total chlorophyll, carotenoids, and carotenoids/chlorophyll ratio are reported between the sun and shade-adapted leaves from other species (Demmig-Adams 1998; Lichtenthaler & Babani 2004, Lichtenthaler et al. 2007). The increased accumulation of chlorophyll in Mikania plants grown in the shade reflects a compensation mechanism to increase the capture of light (Almeida et al. 2005). Chlorophyll is continuously synthesized and degraded (photo-oxidation) in the presence of light, but under conditions of high light intensity, the chlorophyll molecules are more likely to be photo-oxidized, and a balance is established under lower levels of irradiation (Kramer & Kozlowski 1979). The decreases of chlorophyll content and Fv/Fm values are indicators of oxidative stress, as we observed in plants developed under I0. However, in M. laevigata grew under I25 and I50, the lowest chlorophyll content compared to M. glomerata may help in photoprotection due to the reduced light interception, resulting in higher Fv/Fm in this species (Munné-Bosch & Alegra 2000).

Table 1
Anatomical Parameters: Adaxial epidermis thickness (AdE, µm), Abaxial epidermis thickness (AbE, µm), palisade parenchyma (PP, µm), spongy parenchyma (SP, µm), total leaf thickness (TL, µm) in Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker exposed to 0% (I₀), 25% (I₂₅), 50% (I₅₀), and 75% (I₇₅) irradiance interference, at 90 and 150 days after the treatments started (DAT).

Species	Irradiance Interference	90 DAT					150 DAT
		AdE	AbE	PP	SP	TL	AdE	AbE	PP	SP	TL
Mikania	I0	23.27 ± 1.28	21.27 ± 0.63	91.66 ± 2.62	176.35 ± 4.65	312.54 ± 6.08	19.83 ± 0.64	16.47 ± 0.34	72.12 ± 1.38	145.07 ± 2.75	246.20 ± 5.26
glomerata	I25	21.11 ± 1.04	21.40 ± 0.85	90.42 ± 3.58	171.21 ± 5.27	304.14 ± 8.19	17.66 ± 0.52	15.43 ± 0.31	67.30 ± 1.31	137.93 ± 2.66	231.46 ± 4.84
	I50	20.57 ± 1.17	19.99 ± 0.67	94.82 ± 3.02	161.67 ± 5.19	297.06 ± 7.30	17.23 ± 0.44	17.65 ± 0.37	74.61 ± 1.73	137.28 ± 2.81	239.67 ± 5.46
	I75	19.30 ± 0.91	20.41 ± 0.78	87.41 ± 3.91	159.70 ± 7.44	286.82 ± 10.86	15.74 ± 0.52	14.42 ± 0.33	64.47 ± 1.22	143.13 ± 2.90	230.91 ± 4.89
Mikania	I0	33.75 ± 1.80	19.83 ± 0.82	111.66 ± 2.87	192.25 ± 3.61	357.48 ± 4.55	31.89 ± 0.99	16.66 ± 0.33	79.31 ± 1.43	176.39 ± 2.73	295.49 ± 5.52
laevigata	I25	31.04 ± 1.43	20.30 ± 0.69	87.56 ± 3.23	172.37 ± 3.99	311.27 ± 3.98	30.62 ± 0.81	16.89 ± 0.38	74.83 ± 1.39	144.55 ± 2.55	259.21 ± 5.02
	I50	28.80 ± 1.45	20.08 ± 0.64	90.02 ± 2.44	160.68 ± 4.26	299.58 ± 5.33	29.98 ± 0.63	13.83 ± 0.25	55.77 ± 1.03	129.72 ± 2.34	222.70 ± 4.26
	I75	23.11 ± 1.27	19.09 ± 0.61	82.98 ± 2.46	153.73 ± 4.29	278.90 ± 5.37	22.52 ± 0.82	14.46 ± 0.31	66.51 ± 1.59	139.91 ± 2.54	236.40 ± 4.95
ANOVA	Species	**	ns	ns	ns	*	**	*	ns	**	**
	Irradiance	**	ns	**	**	**	**	**	**	**	**
	Species x Irradiance	ns	ns	**	ns	**	**	**	**	**	**

• Data are mean ± SD (n = 5). ns, not significant.
• ^*
  0.05 > P > 0.001.
• ^**
  P < 0.001

Table 2
Anatomical Parameters: stomatal index (SI, %) and stomatal density (SD, stomata/mm²) in Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker exposed to 0% (I₀), 25% (I₂₅), 50% (I₅₀), and 75% (I₇₅) irradiance interference, at 90 and 150 days after the treatments started (DAT).

		90 DAT		150 DAT
Species	Irradiance Interference	SI	SD	SI	SD
Mikania	I0	17.55 ± 0.54	248.68 ± 10.79	18.05 ± 0.32	256 ± 6.48
glomerata	I25	16.75 ± 0.64	248.02 ± 14.19	17.57 ± 0.30	242 ± 6.88
	I50	13.92 ± 0.62	192.46 ± 8.17	16.94 ± 0.31	209 ± 5.40
	I75	12.43 ± 0.47	157.41 ± 6.99	14.90 ± 0.28	154 ± 3.74
Mikania	I0	12.51 ± 0.38	165.34 ± 5.95	14.41 ± 0.29	176 ± 4.84
laevigata	I25	12.62 ± 0.45	178.57 ± 8.08	14.75 ± 0.32	166 ± 4.98
	I50	12.14 ± 0.50	154.10 ± 5.70	12.69 ± 0.28	141 ± 3.70
	I75	11.16 ± 0.36	136.90 ± 5.22	12.29 ± 0.24	123 ± 2.94
ANOVA	Species	**	**	**	**
	Irradiance	**	**	**	**
	Species x Irradiance	**	**	*	**

• Data are mean ± SD (n = 5). ns, not significant.
• ^*
  0.05 > P > 0.001.
• ^**
  P < 0.001

Figure 4
Leaf area (a) and specific leaf area (SLA; b) in Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker, at 90 and 150 days after the treatment started (DAT), grown under sunlight irradiation interference treatments: 0% (I0), 25% (I25), 50% (I50), and 75% (I75). Data shown are the means (± SE) for measurements made on five plants per treatment. Different capital letters indicate significant differences (P < 0.05) among sunlight irradiation interference treatments. Different small letters indicate significant differences (P < 0.05) between species, according to the Tukey test.

Figure 5
Biomass accumulation (g dry mass) of leaves, petiole, stem, and roots of Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker, at 90 (a) and 150 (b) days after the treatment started (DAT), grown under sunlight irradiation interference treatments: 0% (I0), 25% (I25), 50% (I50), and 75% (I75). Data shown are the means (± SE) for measurements made on five plants per treatment. Different capital letters indicate significant differences (P < 0.05) among sunlight irradiation interference treatments. Different small letters indicate significant differences (P < 0.05) between species, according to the Tukey test.

High carotenoids levels under high light conditions are an essential mechanism for photoprotection since it prevents photooxidative damage of chloroplast pigments and avoids the formation of singlet oxygen (Demmig-Adams & Adams 1992, Yamamoto & Bassi 1996, Gonçalves et al. 2001). The total number of molecules of carotenoid per chlorophyll molecule is typically higher in leaves grown in high irradiation environments when compared with leaves of shaded plants, which is in agreement with our results.

In addition to the increased chlorophyll content under shaded conditions, morphological alterations such as higher LA and SLA help plants in light interception process under shaded environments, as observed in Mikania plants under I75 (Artru 2018). In plants developed under I0, the SLA reduction enhance the protection and buffer the damages of excessive solar radiance may cause (Givnish et al. 2004, Matos et al. 2009, Wentworth et al. 2006). The small and dense leaves of plants developed under full sunlight is a morphological mechanism in order to avoid excessive water losses and protection of photosynthetic apparatus against possible photo-oxidative damages caused by excessive solar radiances (Lima Junior et al. 2005).

Lowest SLA values indicate an increased leaf thickness as a result of thicker parenchyma and epidermis under high solar radiances levels. In M. laevigata plants, we observed a higher leaf thickness compared to M. glomerata. This increment in leaf thickness, mainly due to a thicker adaxial epidermis may explain the reduced photoinhibition and higher photosynthesis in plants developed under I25. Epidermis attenuates the UV radiation and allows photosynthetically active radiation (PAR) to diffuse into photosynthetic tissues. For example, Verdaguer et al. (2017) found a thicker adaxial epidermis in plants stimulated by UV-A radiation. The development of a thicker adaxial epidermis, or hypoderm, seems to be a mechanism of protection of palisade parenchyma against the excessive visible light and a protection against leaf wilting when the plant is exposed to high solar radiances levels (Chazdon & Kaufmann 1993). Changes in leaf structure are crucial for plant growth under different environmental conditions (Hanba et al. 2002, Schlüter et al. 2003). The SD and SI in Mikania leaves were also affected by solar radiances levels. The synchronized increases in SD and SI indicated that solar radiances levels stimulated the process of epidermal cell differentiation in stomata and that SD and SI alterations were not caused by reductions in the size or number of epidermal cells (Woodward 1987). The increased SD and SI in plants with the lowest retention of solar radiation flux presumably resulted in an increased g_s and A.

In this study, we observed higher dry matter production under high solar radiances levels in both Mikania species. Similar results were observed in other lianas species, such as Acacia kamerunensis, Loeseneriella rowlandii, and Afrobrunnichia erecta (Toledo-Aceves & Swaine 2008), and Plukenetia volubilis (Cai 2011). Increased biomass accumulation highly depends on the transport of photoassimilates between organs, and patterns of cell division and cell expansion. In this study, high light levels induced A, presumably increasing the production of carbohydrates and dry matter. Our data showed that plants developed under low light availability conditions allocated a greater amount of assimilates to aerial organs in order to reach the light at the upper canopy layer, optimizing the photosynthetic process within an environment where low light might limit photosynthesis (Thompson et al. 1992, Walters et al. 1993). Leaf biomass in both Mikania species was higher in plants grown under higher light conditions, contradicting the results of Boeger et al. (2009), where authors showed that shading stimulated greater leaf biomass production in M. glomerata. In this study, we also observed higher root biomass at full sunlight conditions in both species. This response presumably allows greater absorption of water and nutrients, which may represent a strategy to increase the ability to withstand higher rates of photosynthesis and transpiration in bright environments (Poorter 2001, Mielke & Schaffer 2010).

Our data showed that plants developed under low light intensity showed an increased content of chlorophyll, LA, and SLA. However, A was small when compared to other treatments, presumably resulting in less aboveground dry mass. In both Mikania species, plants developed under shaded environments (I75) showed lower performance than plants grown in other environments. Plants grown under I25 presented higher photosynthesis values than plants grown under I0, and this response may be associated with an increased amount of chlorophyll. Although both species are morphologically similar, M. glomerata and M. laevigata showed distinct responses to irradiance levels. M. glomerata had great biomass production under I0, I25, and I50 with low variation regarding photosynthetic rates. While in M. laevigata plants, the highest photosynthesis rate was observed under I25 and I50, while biomass production was higher under I25. In this species we also observed a higher investment in root growth under higher solar radiances levels (I0 e I25), resulting in a decreased aboveground biomass when compared to M. glomerata. While M. glomerata showed higher SI, SD, LA, and SLA, M. laevigata showed a thicker leaf. In addition to a thicker leaf, caused especially by an increased AdE, the lower chlorophyll content and higher photosynthesis rate observed under I25 and I50 presumably contributed to the reduced photoinibitory effects (Fv/Fm) in this species when compared to M. glomerata, under the same solar radiances level.

Conclusions

We concluded that M. laevigata showed better performance under 25% of retention of solar radiation flux, and not under full sun, contradicting our main hypothesis. We also concluded that plants of both Mikania species show a low capacity of growing under shading conditions. However, under shading both species invested more in shoot biomass, a strategy presumably associate with enhanced growth to reach the canopy more quickly. Although lianas are known as light-dependent plants, the two species presented different responses under the same irradiance conditions. M. laevigata showed better growth under shading of 25% irradiation interference, while M. glomerata grows better from 0 to 50% irradiation interference.

Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Literature cited

• Almeida, S.M.Z., Soares, A.M., Castro, E.M., Vieira, C.V. & Gajego, E.B. 2005. Alterações morfológicas e alocação de biomassa em plantas jovens de espécies florestais sob diferentes condições de sombreamento. Ciência Rural 35: 62-68.
• Artru, S., Lassoisa, L., Vancutsemb, F., Reubensc, B. & Garréa, S. 2018. Sugar beet development under dynamic shade environments in temperate conditions. European Journal Agronomy 97: 38-47.
• Bazzaz, F.A. & Carlson, R.W. 1982. Photosynthetic acclimation to variability in the light environment of early and late successional plants. Oecologia 54: 313-316.
• Bellard, C., Leclerc, C., Leroy, B., Bakkenes, M., Veloz, S., Thuiller, W. & Courchamp, F. 2014. Vulnerability of biodiversity hotspots to global change. Global Ecology and Biogeography 23: 1376-1386.
• Boeger, M.R.T., Espíndola Júnior, A., Maccari Júnior, A., Reissmann, C.B., Alves, A.C.A. & Rickli, F.L. 2009. Variação estrutural foliar de espécies medicinais em consórcio com erva-mate, sob diferentes intensidades luminosas. Floresta 39: 215-225.
• Bolhàr-Nordenkampf, H.R. & Öquist, G. 1993. Chlorophyll fluorescence as a tool in photosynthesis research. In: D.O Hall, J.M.O. Scurlock, H.R. Bolhàr-nordenkampf, R.C. Leegood & S.P. Long (eds.). Photosynthesis and production in a changing environment: a field and laboratory manual. Chapman & Hall, New York, pp. 193-206.
• Caemmerer, V.S. & Baker, N. 2007. The biology of transpiration. From guard cells to globe. Plant Physiology 143: 3.
• Cai, Z.Q. 2011. Shade delayed flowering and decreased photosynthesis, growth and yield of Sacha Inchi (Plukenetia volubilis) plants. Industrial Crops and Products 34: 1235-1237.
• Campos, M.A.A. & Uchida, T. 2002. Influência do sombreamento no crescimento de mudas de três espécies amazônicas. Pesquisa Agropecuária Brasileira 37: 281-288.
• Chazdon, R.L. & Kaufmann, S. 1993. Plasticity of leaf anatomy of two forest shrubs in relation to photosynthetic irradiance acclimation. Functional Ecology 7: 385-394.
• Choudhury, N.K. & Behera, R.K. 2001. Photoinhibition of photosynthesis: role of carotenoids in photoprotection of chloroplast constituents. Photosynthetica 39: 481-488.
• Demmig-Adams, B. 1998. Survey of thermal energy dissipation and pigment composition in sun and shade leaves. Plant & Cell Physiology 39: 474-482.
• Demmig-Adams, B. & Adams, W.W. 1992. Carotenoid composition in sun and shade leaves of plants with different life forms. Plant Cell Environment 15: 411-419.
• DeWalt, S.J., Schnitzer, S.A. & Denslow, J.S. 2000. Density and diversity of lianas along a seasonal tropical forest chronosequence in central Panama. Journal of Tropical Ecology 16: 1-19.
• Dias-Filho, M.B. 1997. Physiological response of Solanum crinitum Lam. to constrasting forest ligth intensity. Pesquisa Agropecuária Brasileira 32: 789-796.
• Gasparetto, J.C., Campos, F.R., Budel, J.M. & Pontarolo, R. 2010. Mikania glomerata Spreng. e M. laevigata Sch. Bip. ex Baker, Asteraceae: estudos agronômicos, genéticos, morfoanatômicos, químicos, farmacológicos, toxicológicos e uso nos programas de fitoterapia do Brasil. Revista Brasileira de Farmacognosia 20: 627-640.
• Gentry, A.H. & Dodson, C. 1987. Contribution of nontrees to species richness of a tropical rain forest. Biotropica 19: 149-156.
• Gerwing, J.J. 2004. Life history diversity among six species of canopy lianas in an old-growth forest of the eastern Brazilian Amazon. Forest Ecology and Management 190: 57-72.
• Givnish, T.J., Montgomery, R.A. & Goldstein, G. 2004. Adaptive radiation of photosynthetic physiology in the Hawaiian lobeliads: light regimes, static light responses and whole-plant compensation points. American Journal of Botany 91: 228-246.
• Gonçalves, C.J.F., Marenco, A.R. & Vieira, G. 2001. Concentrations of photosynthetic pigments and chlorophyll fluorescence of Mahogany and Tonka bean under two light environments. Revista Brasileira Fisiologia Vegetal 13: 149-157.
• Granados, J. & Körner, C. 2002. In deep shade, elevated CO₂ increases the vigor of tropical climbing plants. Global Change Biology 8: 1109-1117.
• Gratani, L. 2014. Plant Phenotypic Plasticity in Response to Environmental Factors. Advances in Botany ID 208747, 1-17.
• Grauel, W.T. & Putz, F.E. 2004. Effects of lianas on growth and regeneration of Prioria copaifera in Darien, Panama. Forest Ecology and Management 190: 99-108.
• Hanba, Y.T., Kogami, H. & Terashima, L. 2002. The effects of growth irradiance on leaf anatomy and photosynthesis in Acer species differing in light demand. Plant Cell Environment 25: 1021-1030.
• Hendry, G.A.F. & Price, A.H. 1993. Stress indicators: chlorophylls and carotenoids. In: G.A.F. Hendry, J.P. Grime (eds.). Methods in comparative plant ecology. Chapman & Hall, New York, pp. 148-152.
• Hsie, B.S., Mendes, K.R., Antunes, W.C., Endres, L., Campos, M.LO., Souza, F.C., Santos, N.D., Singh, B., Arruda, E.C.P. & Pompelli, M.F. 2015. Jatropha curcas L. (Euphorbiaceae) modulates stomatal traits in response to leaf-to-air vapor pressure deficit. Biomass Bioenergy 81: 273-281.
• Ingwell, L.L., Wright S.J., Becklund K.K., Hubbell, S.P. & Schnitzer, A.S. 2010. The impact of lianas on 10 years of tree growth and mortality on Barro Colorado Island, Panama. Journal of Ecology 98: 879-887.
• Kazda, M., Miladera, J. C. & Salzer, J. 2009. Optimisation of spatial allocation patterns in lianas compared to trees used for support. Trees 23: 295-304.
• Kramer, T. & Kozlowski, T. 1979. Physiology of woody plants. Academic, New York
• Kraus, J.E. & Arduin, M. 1997. Manual básico de métodos em morfologia vegetal. EDUR, Seropédica.
• Kurzel, B.P., Schnitzer, S.A. & Carson, W.P. 2006. Predicting Liana crown location from stem diameter in three Panamanian Lowland Forests. Biotropica 38: 262-266.
• Lambers, H., Chapin, S.T., III & Pons, T.J. 1998. Plant Physiological Ecology. Springer-Verlag, Berlin.
• Laurance, W.F., Perez-Salicrup, D., Delamonica, P., Fearnside, P.M., D’Angelo, S., Jerozolinski, A., Pohl, L. & Lovejoy, T.E. 2001. Rain forest fragmentation and the structure of Amazonian liana communities. Ecology 82: 105-116.
• Lichtenthaler, H.K., Ač, A., Marek, M.B., Kalina, J. & Urban, O. 2007. Differences in pigment composition, photosynthetic rates and chlorophyll fluorescence images of sun and shade leaves of four tree species. Plant Physiology and Biochemistry 45: 577-588.
• Lichtenthaler, H.K. & Babani, F. 2004. Light adaptations and senescence of the photosynthetic apparatus. Changes in pigment composition, chlorophyll fluorescence parameters and photosynthetic activity. In: G.C. Papageorgiou, Govindjee (eds.). Chlorophyll Fluorescence: A Signature of Photosynthesis. Springer, Dordrecht, pp. 713-736.
• Lima Junior, E.C., Alvarenga, A.A., Castro, E.M., Vieira, C.V. & Oliveira, H.M. 2005. Trocas gasosas, características das folhas e crescimento de plantas jovens de Cupania vernalis Camb. submetidas a diferentes níveis de sombreamento. Ciência Rural 35: 1092-1097.
• Lima, N.P., Biasi, L.A., Zanette, F. & Nakashima, T. 2003. Produção de mudas por estaquia de duas espécies de guaco. Horticultura Brasileira 21: 106-109.
• Matos, F.S., Wolfgramm, R., Cavatte, P.C., Villela, F.G., Ventrella, M.C. & Matta, F.M. 2009. Phenotypic plasticity in response to light in the coffee tree. Environment Experimental Botany 67: 421-427.
• Metzger, J. P. 2009. Conservation issues in the Brazilian Atlantic forest. Biological Conservation 142: 1138-1140.
• Mielke, M.S. & Schaffer, B. 2010. Photosynthetic and growth responses of Eugenia uniflora L. seedlings to soil flooding and light intensity. Environment Experimental Botany 68: 113-121.
• Munné-Bosch, S. & Alegra, L. 2000. Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus offcinalis plants. Planta 210: 925-931.
• Myers, N., Mittermeier, R.A., Mittermeier, C.G., Fonseca, G.A.B. &Kent, J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.
• Nabe-Nielsen, J. 2002. Growth and mortality rates of the liana Machaerium cuspidatum in relation to light and topographic position. Biotropica 34: 319-322.
• Phillips, O.L., Martínez, R.V., Mendoza, A.M., Baker, T. R. &Vargas, P.N. 2005. Large lianas as hyperdynamic elements of the tropical forest canopy. Ecology 86: 1250-1258.
• Phillips, O.L., Martínez, R.V., Arroyo, L., Baker, T.R., Killeen, T., Lewis, S.L., Malhi, Y., Mendoza, A.M., Neill, D., Vargas, P.N., Alexiades, M., Cerón, C., Di Fiori, A., Erwin, T., Jardim, A., Palacios, W., Saldias, M. & Vinceti, B. 2002. Increasing dominance of large lianas in Amazonian forests. Nature 418: 770-774.
• Poorter, L. 2001. Light-dependent changes in biomass allocation and their importance for growth of rain forest tree species. Functional Ecology 15: 113-123.
• Putz, F.E. 1984. The natural history of lianas on Barro Colorado Island, Panama. Ecology 65: 1713-1724.
• Rezende, C.L., Sacarano, F.R., Assad, E.D., Joly, C.A., Metzger, J.P., Strassburg, B.B.N., Tabarelli, M., Fonseca, G.A. & Mittermeier, R.A. 2018. From hotspot to hopespot: An opportunity for the Brazilian Atlantic Forest. Perspectives in Ecology and Conservation 16: 208-214.
• Ribeiro, R.V., Souza, G.M., Oliveira, R.F. & Machado, E.C. 2005. Photosynthetic responses of tropical tree species from different sucessional groups under contrasting irradiance conditions. Revista Brasileira de Botânica 28: 149-161.
• Ricketts, T.H., Dinerstein, E., Boucher, T., Brooks, T.M., Butchart, S.H.M., Hoffmann, M., Lamoreux, J.F., Morrison, J., Parr, M., Pilgrim, J.D., Rodrigues, A.S.L., Sechrest, W, Wallace, G.E., Berlin, K., Bielby, J., Burgess, N.D., Church, D.R., Cox, N., Knox, D., Loucks, C., Luck, G.W., Master,L.L., Moore, R., Naidoo, R., Ridgely, R., Schatz, G.E., Shire, G., Strand, H., Wettengel,W., & Wikramanayake,E. 2005. Pinpointing and preventing imminent extinctions. Proceedings of the National Academy of Sciences of the United States of America 102: 18497-18501.
• Sanches, M.C. & Válio, I.F.M. 2002. Seedling growth of climbing species from a southeast Brazilian tropical forest. Plant Ecology 154: 51-59.
• Schnitzer, S.A. 2005. A mechanistic explanation for global patterns of liana abundance and distribution. The American Naturalist 166: 262-276.
• Schnitzer, S.A. & Bongers, F. 2002. The ecology of lianas and their role in forests. Trends in Ecology & Evolution 17: 223-230.
• Schnitzer, S.A. & Bongers, F. 2011. Increasing liana abundance and biomass in tropical forests: emerging patterns and putative mechanisms. Ecology Letters 14: 397-406.
• Schnitzer, S.A. & Carson, W.P. 2010. Lianas suppress tree regeneration and diversity in treefall gaps. Ecology Letters 13: 849-857.
• Schnitzer, S.A., Mangan, S.A., Dalling, J.W., Baldeck, C.A., Hubbell, S.P., Ledo, A., Muller-Landau, H., Tobin, M.F., Aguilar, S., Brassfield, D., Hernandez, A., Lao, S., Perez, R., Valdes, O. & Yorke, S.R. 2012. Liana Abundance, Diversity, and Distribution on Barro Colorado Island, Panama. Plos One 7 e52114.
• Schlüter, U., Muschak, M., Berger, D. & Altmann, T. 2003. Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1-1) under different light regimes. Journal Experimental Botany 54: 867-874.
• Thompson, W.A., Huang, L.K. & Kriedemann, P.E. 1992. Photosynthetic response to light and nutrients in sun-tolerant and shade-tolerant rainforest trees. II. Leaf gas exchange and component processes of photosynthesis. Australian Journal Plant Physiology 19: 19-42.
• Toledo-Aceves, T. & Swaine, M.D. 2008. Biomass allocation and photosynthetic responses of lianas and pioneer tree seedling to light. Acta Oecologica 34: 38-49.
• Toledo, A.C.O., Hirata, L.L., Buffon, M.C.M., Miguel, M.D. & Miguel, O.G. 2003. Fitoterápicos: uma abordagem farmacotécnica. Revista Lecta 21: 7-13.
• van der Heijden, G.M., Schnitzer, S.A., Powers, J.S. & Phillips, O.L. 2013. Liana impacts on carbon cycling, storage and sequestration in tropical forests. Biotropica 45: 682-692.
• van der Heijden, G.M.F., Powers, J.S. & Schnitzer, S.A. 2015. Lianas reduce carbon accumulation and storage in tropical forests. Proceedings of the National Academy of Sciences of the United States of America 112: 13267-13271.
• Verdaguer, D., Jansenb, M.A.K., Llorensa, L., Morales, L.O. & Neugartd, S. 2017. UV-A radiation effects on higher plants: Exploring the known unknown. Plant Science 255: 72-81.
• Walters, M.B., Kruger, E.L. & Reich, P.B. 1993. Growth, biomass distribution and CO₂ exchange of northern hardwood seedlings in high and low light: relationships with successional status and shade tolerance. Oecologia 94: 7-16.
• Wentworth, M., Murchie, E.H., Gray, J.E., Villegas, D., Pastenes, C., Pinto, M. & Horton, P. 2006. Differential adaptation of two varieties of common bean to abiotic stress. Journal Experimental Botany 57: 699-709.
• Woodward, F.I. 1987. Stomatal numbers are sensitive to increases in CO₂ from pre-industrial levels. Nature 327: 617-618.
• Wright, S.J., Calderon, O., Hernandez, A. & Paton, S. 2004. Are lianas increasing in importance in tropical forests? A 17- year record from Panama. Ecology 85: 484-489.
• Yamamoto, H.Y. & Bassi, R. 1996. Carotenoids: localization and function. In: D.R. Ort, C.F. Yocum & I.F. Heichel (eds.). Oxygenic Photosynthesis: The Light Reactions. Advances in Photosynthesis and Respiration, v. 4. Springer, Dordrecht.
• Zhu, H., Xu, Z.F., Wang, H. & Li, B.G. 2004. Tropical rain Forest fragmentation and its ecological and species diversity changes in southern Yunan. Biodiversity & Conservation 13: 1355-1372.
• Zhu, S.-D. & Cao, K.-F. 2009. Hydraulic properties and photosynthetic rates in co-occurring lianas and trees in a seasonal tropical rainforest in southwestern China. Plant Ecology 204: 295-304.

Supplementary material

Supplementary material Table S1
Monthly climatological conditions in the study site during the experiment.

Month	Days of treatment	Maximum temperature (°C)	Minimum temperature (°C)	Average temperature (°C)	Precipitation (mm)
May	0	25.79	13.31	19.56	6.21
June	30	26.42	13.72	20.07	0.33
July	60	27.43	11.80	19.61	0.00
August	90	29.76	15.41	22.60	2.11
September	120	30.10	15.32	22.72	0.89
October	150	30.91	18.96	24.94	5.56

Supplementary material Table S2
The results of a two-way analysis of variance (ANOVA) with the species (S) and irradiance (I) as fixed factors with the interaction (S × I) is shown at each analyzed parameter.

Parameter	DAT
		60			90			120			150
	S	I	S x I	S	I	S x I	S	I	S x I	S	I	S x I
E	ns	ns	ns	ns	*	ns	**	**	ns	ns	**	**
gs	ns	*	ns	*	**	ns	*	**	ns	*	**	*
A	ns	*	ns	ns	**	ns	ns	**	ns	ns	**	*
iWUE	ns	ns	ns	**	ns	ns	**	*	**	**	**	*
Chlorophyll	*	**	ns	ns	**	ns	*	**	ns	**	**	ns
Carotenoids	*	**	ns	ns	**	ns	*	**	ns	**	**	ns
Car/Chl	ns	**	ns	*	**	ns	*	**	*	ns	**	ns

• Data are mean ± SD (n = 5). ns, not significant.
• ^*
  0.05 > P > 0.001.
• ^**
  P < 0.001

Supplementary material Table S3
Average temperature (T, ºC), relative humidity (RH, %) and vapor pressure deficit (VPD, kPa) in 0% (I0), 25% (I25), 50% (I50), and 75% (I75) irradiation interference.

Days of treatments		I0			I25			I50			I75
	T	RH	VPD	T	RH	VPD	T	RH	VPD	T	RH	VPD
60	14.8	69.0	0.5	14.9	69.5	0.5	14.9	70.5	0.5	14.7	71.5	0.5
90	23.9	53.0	1.4	23.1	51.5	1.4	22.1	51.5	1.3	21.2	51.5	1.2
120	28.5	17.7	3.2	27.8	18.7	3.0	26.8	18.2	2.9	25.8	18.0	2.7
150	33.1	31.7	3.5	32.0	33.0	3.2	30.8	31.2	3.0	29.4	31.0	2.8

Supplementary material Figure S1
Diurnal course of relative humidity, ambient temperature, and the photosynthetic photon flux density (PPFD) at 60 (a, e, i), 90 (b, f, j), 120 (c, g, k), and 150 DAT (d, h, l). Treatments: 0% (I0), 25% (I25), 50% (I50), and 75% (I75) of irradiance interference.

Edited by

Associate Editor: Wagner de Melo Ferreira

Publication Dates

• Publication in this collection
  22 Nov 2021
• Date of issue
  2021

History

• Received
  22 June 2020
• Accepted
  19 July 2020