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

(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.


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, SciELO Preprints -This document is a preprint and its current status is available at: https://doi. org/10.1590/2236-8906-74/2020 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 SciELO Preprints -This document is a preprint and its current status is available at: https://doi. org/10.1590/2236-8906-74/2020 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, see 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 figure 1). 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.
The measurements were made under ambient conditions of radiation, [CO2], and air temperature.
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.
SciELO Preprints -This document is a preprint and its current status is available at: https://doi. org/10.1590/2236-8906-74/2020 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: 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 cm 2 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 (dm 2 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.
SciELO Preprints -This document is a preprint and its current status is available at: https://doi. org/10.1590/2236-8906-74/2020 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).

Results
Gas exchange -The solar radiation level significantly affected stomatal conductance (gs) and 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 gs and E. The increased air temperature and VPD observed at 150 DAT presumably caused the reduction in gs, 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. SciELO Preprints -This document is a preprint and its current status is available at: https://doi. org/10.1590/2236-8906-74/2020 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,

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. SciELO Preprints -This document is a preprint and its current status is available at: https://doi. org/10.1590/2236-8906-74/2020 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.

Authors contribution
Daniele Ribeiro Contin: carried out the experiments, collected and analyzed data and prepared the manuscript.
Eduardo Habermann: prepared the manuscript and contributed to the revision of the manuscript.
Carlos Alberto Martinez: supervised the study and contributed to the revision of the manuscript.