Area of study

The study was conducted in three localities in the municipality of Palmira, department of Valle del Cauca: the first in the Mario González Aranda Farm of the National University of Colombia, Palmira campus. The climate in this region is classified as tropical dry forest (^{Henríquez et al., 2005}) with average annual temperature and precipitation of 24 °C and 1 020 mm respectively and an altitude of 1 000 masl. The soil classification is Clayey (Cl) with > 19.80% sand, 0.08 of organic matter and a pH of 6.9.

The second and third localities were located in the village of La Veranera, corregimiento of Toche. The climatic classification of the area is very humid montane forest (^{Henríquez et al., 2005}) with average annual temperature and precipitation of 15 °C and 1 800 mm, respectively. The height masl varies between 1 500 and 2 000. The classification of the soil at 1 500 and 2 000 masl is Loamy (L) with >20.32% clay, organic matter 0.10 and pH 6 and in Sandy loamy (SL) with >36.32% clay, organic matter 0.26 and pH 6.1, respectively.

Experimental design, data collection and forage species

The forage species evaluated were Star (Cynodon plectostachyus K. Schum), Kikuyu (Pennisetum clandestinum Hochst. ex Chiov), Toledo (Urochloa brizantha Hochst. ex A. Rich. CIAT 26110), Humidicola (Urochloa humidicola Rendle. Schweick. CIAT 26159), Mulato II (Urochloa hibrido, CIAT 36087), Tanzania (Megathyrsus max Jacq) and Centrosema molle (Mart. ex Benth CIAT 15160). Twenty-eight plots of 8 m² (4*2 m) were established at 1 000, 1 500 and 2 000 masl.

Data collection was carried out by randomly selecting three healthy plants, in each of the plots of the six grasses and one legume, in the rainy and dry seasons in 2019 every 20 days between 8:00 am and 10:00 am. For each beginning of the season, a uniformity cut was made according to those suggested by ^{Toledo and Schultze (1982)}. Readings of photosynthesis rate (TA) μmol CO₂ m^-2 s^-1, stomatal conductance (G) mol m^-2 s^-1, transpiration rate (E) mmol m^-2 s^-1 and internal CO₂ (Ci) μmolmol^-1 were made using the Lcpro+ portable photosynthetic measurement analyzer, manufactured by the company ADC Bio-Scientific in the UK.

Statistical analysis

Data collected during readings were analyzed using the general linear program (GLM) of the statistical program SAS^® V.9.3 (^{SAS Institute Inc., 2011}). Duncan’s test (p≤ 0.05) was used for comparison of treatment means.

Gas exchange

The statistical analysis of the data indicates that the TA by species and season presented a variation between 35.95 and 15.13 μmol CO₂ m^-2 s^-1 and 30.94 and 28.97 μmol CO₂ m^-2 s^-1 respectively, Table 1. The highest TA was observed in the species of Star, Mulato II, Toledo and Tanzania (Table 1). This is similar to those reported by ^{Silva et al. (2012)} in cultivars of the genus Megathyrsus, Cynodon and Urochloa for the same seasons in Brazil.

These cultivars would have a better capacity to withstand long periods of drought and rain, in addition to being more efficient to intercept the incident energy available in these seasons by developing adaptation mechanisms by modifying the physical nature of their roots and leaves in order to regulate the entry and exit of water and CO₂ (^{Peters et al., 2011}; ^{Silva et al., 2012}). The TA for Kikuyu grass was similar in the two seasons evaluated (Table 1), which would indicate its adaptability to tropical climates and its ability to photosynthesize in temperature ranges different from the one in which it is commonly found (^{Álvarez et al., 2008}).

The legume of the species Centrosema molle presented the lowest TA with 15.13 μmol CO₂ m^-2 s^-1 in both seasons. This value is above that reported by ^{Xiong et al. (2017)}, in leguminous forages of Trifolium repens, where the observed value was 11.65 and 6.55 μmol CO₂ m^-2 s^-1 in the same seasons. The differences between these two legume species are possibly due to the environmental conditions in which they were established, allowing Centrosema molle to develop adaptation mechanisms conducive to the tropics.

Grasses have specific physiological and morphological characteristics that provide specific adaptation for their growth and quality. However, these undergo modifications in morphology, in yield and quality when there are changes in the climatic conditions, where temperature, solar radiation (quantity and quality), rainfall and its distribution are the components that most determine tropical conditions (^{Del Pozo, 2002}).

The species of Star and Centrosema molle presented the highest and lowest TA, respectively (Table 1). This is due to the fact that Star, by having a C4 photosynthetic pathway, presents a better adaptation to high temperatures and a low concentration of atmospheric CO₂ compared to grasses of C3 photosynthetic pathway (^{Loomis and Amthor, 1999}; ^{Taylor et al., 2012}). The physiological explanation for this adaptation mechanism is that the C4 photosynthetic pathway requires less Rubisco, so consequently and importantly, less foliar nitrogen (N) per leaf area unit for rapid photosynthesis.

In addition, they have a water distribution structurally different from that of C3 plants. Allowing C4 grasses to use water and nitrogen efficiently to achieve high growth rates, provided temperatures are adequate (^{Sierra, 2005}; ^{Crush and Rowarth, 2007}). On the other hand, temperate grasses and tropical and temperate legumes, such as the species Centrosema, use the C3 pathway to perform photosynthesis. This pathway has a highly sophisticated enzyme complex called ribulose 1,5-bisphosphate carboxylase (Rubisco), which has an affinity for oxygen (^{Taiz and Zeiger, 2010}).

This leads to a lower rate of photosynthesis because the plant has to expend a reasonable amount of energy and nutrients to eliminate O₂. It is considered that the losses resulting from photorespiration, observed in C3-type plants, cause a decrease between 20 and 70% of photosynthesis (^{Machado, 1988}, ^{Bonan, 2015}).

The evaluated grasses presented different TAs between them (Table 1). This is possibly due to the fact that there are structural and biochemical variations in CO₂ fixation among C4-pathway plants (^{Coombs, 1988}, ^{Crush and Rowarth, 2007}). ^{Stitt et al. (2010)} indicate that there are three types of C4 systems, one of them is represented by plants that present Kranz-type anatomy, characterized by mesophyll parenchymal cells organized around the vascular bundle cells.

The other system is monomorphic, which occurs in a single cell and has chloroplasts with decarboxylases, as well as the enzyme Rubisco. The third C4 system is the dimorphic system, characterized by having two types of chloroplasts, with different functionalities and biochemical processes, which allow spatial compartmentalization within a single cell (^{Offermann et al., 2011}). As for Gs and E, the highest values were observed for Centrosema molle, Star, Toledo, Tanzania, Mulato II and Humidicola (Table 1). ^{Atencio et al. (2014)} reported a higher Gs and E in Urochloa Humidicola, Mulato II, Toledo and Mombaza as well as a high value in the TA of these species, which presents similarity with what was reported in this work.

This would indicate that these species would have a full activity of photosynthetic processes and a better hydration condition. On the other hand, this type of results in C4-type plants would demonstrate that an increase in atmospheric CO₂ would be beneficial to increase biomass production, as well as to reduce stomatal conductance and transpiration even when no effect on the instantaneous rate of photosynthesis is observed (^{Sánchez et al., 2000}; ^{Pritchard and Amthor, 2005}). The species Centrosema molle, which is a C3-type plant, presented high values for Gs and E (Table 1).

These results can be compared with those of ^{Guenni et al. (2018)}, which report a Gs of 0.8 mol m^-2 s^-1 and E of 7.2 mmol m^-2 s^-1 with a TA of 14.9 μmol CO₂ m^-2 s^-1. This may indicate that this species grew in the midst of a high concentration of atmospheric CO₂, causing an effect on the increase of the stomatal opening as an adaptation to these high levels of CO₂ (^{Sánchez et al., 2000}). The lowest values of Gs and E were observed in the species Kikuyu with 0.82 mol m^-2 s^-1 and 4.95 mmol m^-2 s^-1 respectively. According to ^{Pereira et al. (2012)}, reduction in CO₂ assimilation rates and stomatal conductance are associated with low water potential in leaves or the reduction in water content in soil.

In addition, plants when subjected to water stress conditions reduce the efficiency of solar radiation use, which also affects photosynthesis (^{Taylor et al., 2012}; ^{Gonçalves et al., 2015}). The variable Ci showed a wide variation between the species evaluated (Table 1). Centrosema molle and Kikuyu presented the highest value with 272.81 and 199.46 μmolmol^-1 respectively. This would indicate that these two forage species have a lower fixation of Ci at the time of photosynthesis due to the photorespiration process (^{Tolbert, 1980}; ^{Ogren, 1984}), especially Centrosema molle which, by having a C3 metabolism, is favored with the increase of this gas in the active site of Rubisco (^{Simões et al., 2009}).

The high Ci in Kikuyu, which has a C4 metabolism, is possibly due to the fact that it has developed adaptation mechanisms for environments of warm climate (^{Taiz and Zeiger, 2010}). The other species evaluated presented relatively low Ci values, confirming their condition as C4 plants, Table 1 (^{Sierra, 2005}; ^{Da Matta et al., 2001}; ^{Dias, 2002}). Recent comparative studies of grasses have indicated that photosynthesis of C4 species is an adaptation to low atmospheric CO₂ and open habitats, evolving at high temperatures and allowing the colonization of drier and seasonal subtropical environments with which they would have a greater efficiency in water use compared to C3 species (^{Taylor et al., 2012}; ^{Osborne and Freckleton, 2009}).

Photosynthesis and temperature (leaf, chamber and environment) by species and altitude

Table 2 shows the general averages of TA, leaf temperature (LT), chamber temperature (CT) and air temperature (AT) for each species according to altitude.

It was observed that the Star grass presented the highest TA at 1000 masl, when LT was 45 °C (Table 2). This is possibly because plants with C4 photosynthetic pathways better adapt to higher temperatures, which induce higher phosphoenolpyruvate carboxylase (PEPC) activity in this type of species (^{Tolbert, 1980}; ^{Ogren, 1984}). ^{Labate et al. (1990)} indicate that photosynthetic rates in some grasses increase as temperatures increase. The cultivars Mulato II, Toledo and Humidicola obtained similar TA and temperatures, at the three altitudes.

This would indicate that they respond to an increase in temperature by increasing TA. An important aspect of temperature refers to its fluctuation, both throughout the day and throughout the year since each time it varies, the plant must adapt (^{Vieira and Mochel, 2010}). In a study conducted by ^{Dias (2002)} in Urochloa sp., he observed that TA increased as temperature increased. Tanzania had a TA between 31 and 34 μmol CO₂ m^-2 s^-1 with temperatures ≥28 °C at the three altitudes (Table 2).

This is similar to what was reported by ^{Mello et al. (2001)} in the genus Megathyrsus, where they observed a high TA with 34.57 μmoles CO₂ m^-2 s^-1 at an optimal temperature around 35 °C. The TA of Kikuyu grass varied between 31.6 and 27.4 μmoles CO₂ m^-2 s^-1 with temperatures between 36.9 °C and 29.5 °C. This coincides with what was reported by ^{Wilen and Holt (1996)} in the species Pennisetum clandestinum, in sub-warm environments, where the TA increased between 25 °C and 40 °C.

The authors indicate that this species can continue with photosynthesis at higher rates than other C4 species as temperatures decrease in autumn and winter, demonstrating its adaptability in different environments In Centrosema molle no differences in TA were observed between altitudes, although temperatures were different in each (Table 2). A small reduction in TA occurred when the temperature was greater than 40 °C, this could be due to the deactivation of the enzyme Rubisco that controls photosynthesis (^{Crafts and Salvucci, 2000}). Slight decreases in TA above 35 °C suggest that this species better adapts to warm climates (^{Baligar et al., 2010}).