Ecophysiology of two tropical species in an abandoned eucalypt plantation: effect of plant litter removal and seasonality

The effect of the removal of plant litter on photosynthetic variables (gas exchanges, chlorophyll a luorescence, and content of photosynthetic pigments) of the tropical species Xylopia sericea A. St.-Hil. and Siparuna guianensis Aubl. was evaluated in an abandoned plantation of eucalypt (Corymbia citriodora (Hook.) K.D. Hill & L.A.S. Johnson (Myrtaceae)). The study was conducted at the União Biological Reserve, Rio de Janeiro State, Brazil during the rainy and dry periods, after ive years of litter removal. The removal of plant litter did not inluence the ecophysiological responses of the species. There was however signiicant seasonal variation. During the dry period, intercellular CO 2 concentration (C i ), transpiration (E), and stomatal conductance (g s ) were decreased, while intrinsic eficiency of water use (IWUE), non-photochemical quenching (NPQ), and carotenoid values increased, suggesting a protective strategy against stress. Nevertheless, the values for F v /F m (maximum quantum eficiency) and F m /F o (ratio of luorescence yields for open and closed states) indicated that even during the dry period there was no reduction in photochemical activity in these species. Only S. guianensis exhibited a reduced net photosynthetic rate (A) during the dry period. The data indicated that X. sericea was photosynthetically more eficient under conditions of low water availability and that a 5-year period of plant litter removal failed to produce differences in ecophysiological processes in the species analyzed.


Introduction
The Atlantic Forest in Brazil, which once covered more than one million square kilometers, has been reduced to 12% of its original area (RIBEIRO et al., 2009).Given the current state of degradation of this biome, we are confronted with the need to conserve and make sustainable use of its remaining stands, expand native forest plantations, connect fragments, and restore abandoned and degraded areas.With the objective of minimizing pressures on native forests and recovering degraded areas, all while meeting increasing demands for wood and bioenergy; many countries have begun expanding the systematic planting of exotic tree species with rapid initial growth patterns (FEYERA et al., 2002;FONSECA et al., 2009).
Studies have demonstrated that eucalypt plantations can facilitate natural regeneration, thus helping to restore native forests (FEYERA et al., 2002;FERREIRA et al., 2007;NÓBREGA et al., 2008).Some species of eucalypt, however, such as Corymbia citriodora (Hook.)K.D. Hill & L.A.S. Johnson (Myrtaceae), have been considered harmful due to their allelopathic effects (NISHIMURA et al., 1984;ZHANG;FU, 2010).In addition, eucalypt leaf litter is considered to be of low nutritional quality (VILLELA et al., 2004) due to the elevated C/N ratio, which interferes with decomposition rates and nutrition cycling.
Plant litter is essential for the introduction of nutrients into the soil-vegetation system, and is thus an important factor in healthy forest ecosystem functioning (CHAPIN III et al., 2002;PRESCOTT et al., 2004;KNORR et al., 2005).Plant litter is a determinant in the dynamics and structure of plant communities, because of its effects on germination and plant establishment (XIONG; NILSSON, 1999).The plant litter layer protects the soil against erosion, leaching, and sudden changes in temperature and humidity (MO et al., 2003;SAYER, 2006;SANGHA et al., 2006), as well as furnishing a habitat and substrate for soil fauna (ATTIGNON et al., 2004) and decomposition microorganisms (RUF et al., 2006).
In southeastern Brazil, extensive tracts of the Atlantic Forest have been substituted by monoculture tree species (SUGUITURU et al., 2011).Brazil is currently the world leader in total occupied area of eucalypt plantations (GIT FORESTRY CONSULTING, 2009).The União Biological Reserve (REBIO União), located in the southeastern region of Brazil, has abandoned plantations of C. citriodora in which native species are regenerating in understory.However, studies indicate that these plants regenerate very slowly and have low species diversity (EVARISTO, 2008).For example, Xylopia sericea (Annonaceae) and Siparuna guianensis (Siparunaceae), both in the initial stage of ecological succession, are among the native species Ecophysiology of tropical species in an eucalypt plantation that occur in greatest abundance (EVARISTO et al., 2011).An experiment, involving manipulation of plant litter over the long term, was initiated in this reserve in 2004, and it demonstrated that the plant litter functions as a physical barrier against seed germination, while the removal of plant litter generally enhances the recruitment and survival of native species seedlings (RIBEIRO, 2007).On the other hand, in the same area studied, D.M. Villela (unpublished data) obtained results attesting to a negative effect related to the removal of plant litter, with soil showing lower C and N values and C/N ratio when compared with the controls.
Just as with nutrient concentration, water availability of the soil may limit the establishment and productivity of the plants (CAVENDER-BARES; BAZZAZ, 2004).Litter plays an important role in water conservation during dry conditions (FOWLER, 1986;1988).Water stress can affect the photosynthetic process through stomatal and non-stomatal effects.The stomatal effect, considered a primary event, leads to a lower concentration of internal CO 2 , reducing its assimilation in chloroplasts (FARIA et al., 1996).The non-stomatal effects refer to perturbations in photochemical (BAKER, 1993) and biochemical (LU; ZANG, 1999) processes.
Studies focusing on the management of plant litter over long periods are in their initial phases (WOOD et al., 2009;SAYER;TANNER, 2010) and require the consideration of ecophysiological aspects to better understand the resultant nutritional adjustments and/ or dei ciencies in different types of soils.With this in mind, the objectives of this work were: (1) to examine the effect of plant litter removal on the ecophysiological attributes of the species X. sericea and S. guianensis in an abandoned plantation of eucalypt (C.citriodora), and (2) to examine the occurrence of seasonal variations among the ecophysiological attributes and their relation to plant litter removal.

Study area, plant material, and sampling period
The União Biological Reserve, Rio de Janeiro, Brazil (22º27'S and 42º02'W) consists of 2,550 ha, with 2,200 ha of coastal lowland and submontane Atlantic Forest classii ed as dense ombrophilous forest (VELOSO et al., 1991).In addition, approximately 220 ha are covered with eucalypt (C.citriodora) plantations of different ages (MMA-ICMBIO, 2008).The climate is tropical humid with an average annual temperature of 25°C, rainfall on the order of 1,620 mm year -1 , with 75% of rainfall concentrated in the period of October to March (Figure 1).This study was conducted on plots located in an 11.44-ha eucalypt plantation, with spacing of 3 m x 3 m age of 41 years, which had received no forestry management intervention since 1996.According to Evaristo et al. (2011), the density of individuals per hectare was 1,010 with a total of 17 species (X.sericea, S. guianensis, Guatteria campestris R.E.Fr., Piptocarpha macropoda (DC.) Baker, Cybistax antisyphilitica (Mart.)Mart., Jacaranda puberula Cham., Sparattosperma leucanthum (Vell.)K.Schum., Erythroxylum pulchrum A.St.-Hil., Lacistema pubescens Mart., Licaria ).The photosynthetic photon lux density (PPFD) was 196.9 ± 49.9 μmol m -2 s -1 during the rainy period and 178.9 ± 45.4 μmol m -2 s -1 during the dry period, while the average air temperature (understory level) was 32.1 ± 0.8°C during the rainy period and 27.5 ± 0.5°C during the dry period.In August 2004, six plots measuring 5 m x 20 m were randomly demarcated.In three of the plots, the plant litter was maintained (control treatment), and in the other three plots, the plant litter layer was removed every 15 days (removal treatment).
The tree species X. sericea and S. guianensis occur naturally with large numbers of individuals in the understory of this plantation in both treatment areas (EVARISTO, 2008).In total, nine individuals of X. sericea (1.5-2.0 m) and S. guianensis (1.5-2.0 m) were selected from each treatment (three individuals per plot).Gas exchange, chlorophyll a luorescence, and level of photosynthetic pigments in young leaves (totally expanded and collected from the second node) were measured twice during the rainy period (November 2009 and February 2010) and the dry period (July 2010 and September 2010).The results for the control treatment collection are from the data collected by Lage-Pinto et al. (2012).

Gas exchange
Measurements were determined with a portable photosynthesis system, model CIRAS 2 (PP Systems International, Inc. Amesbury, MA, USA) with the use of a leaf cuvette with an area of 1.7 cm 2 .LED unit (PLC6 (U)) was used for light control.Data were collected at ambient conditions of temperature (28 ± 3ºC) and relative humidity (70%).Photosynthetic light response curves were determined for PPFD between 0 and 2,000 μmol m -2 s -1 at [CO 2 ] of 380 μmol mol -1 .All gas exchange measurements were taken after steady rates of photosynthesis and transpiration were observed, usually within 15 to 25 min after exposure to an altered light intensity.The A/PPFD light response curve was used to determine light saturated photosynthesis for both species (700 μmol m -2 s -1 for X. sericea and 1,100 μmol m -2 s -1 for S. guianensis).After determination of light saturated photosynthesis, the following parameters were obtained: net photosynthetic rate (A), intercellular CO 2 concentration (C i ), stomatal conductance (g s ), and transpiration (E).The intrinsic water use eficiency (IWUE) was calculated as the ratio A/g s .Measurements were taken between 8:00 and 10:30 AM during the period of maximum stomatal conductance.Each measurement was performed on two fully expanded mature leaves per individual.

Chlorophyll a luorescence
Chlorophyll a luorescence was measured in situ between 11:00 AM and 12:00 PM with the use of a portable modulated luorometer, model FMS2 (Hansatech Instruments, Ltd, UK).The measurements were done on the same leaves used for gas exchange analysis.After 30 min of adaptation to the dark (with the use of leaf clips), the leaves were exposed to red light modulated at low intensity (approximately 6 mmol m -2 s -1 at 660 nm) followed by a pulse of 0.8 s of actinic light (6,000 µmol m -2 s -1 ), modiied from Genty et al. (1989) and Van Kooten and Snel (1990).The following chlorophyll a luorescence emission parameters were determined: F o (minimum luorescence), F m (maximum luorescence), and F v (variable luorescence), F v /F m (maximum quantum eficiency of photosystem II), qL (photochemical quenching-Kramer lake model), NPQ (non-photochemical quenching) and F m /F o (the ratio of luorescence yields for open and closed states).

Photosynthetic pigments
Three leaf discs were removed from the nine plants evaluated (mature leaves) of each treatment area and their pigments were extracted with 5 mL of dimethyl sulfoxide (DMSO).After ive days, the extract was analyzed in a spectrophotometer (480, 649, and 665 nm).Chlorophyll a, chlorophyll b, and carotenoids Ecophysiology of tropical species in an eucalypt plantation were measured according to Wellburn (1994).The concentrations of total chlorophylls (chlorophyll a+b), chlorophyll a/b, and total chlorophylls/carotenoids were calculated.All laboratory procedures were carried out under low light conditions.

Statistical analyses
The ecophysiological data were separated into two groups, one for the rainy period (November 2009 and February 2010) and one for the dry period (July 2010 and September 2010), resulting in a sample number of 18 for each treatment area.The results of the gas exchange, chlorophyll a luorescence, and level of photosynthetic pigments were subjected to ANOVA for three factors, and the means were compared with the Tukey test.

Results
The variables related to gas exchange did not show signiicant differences between the control and treatment with plant litter removal areas for X. sericea or S. guianensis (Figure 2).However, there was seasonal variation; in the dry period, the variables C i , g s , and E (Figures 2B, 2C, and 2E) showed lower values, while IWUE (Figure 2E) exhibited higher values for both species.Only S. guianensis showed a signiicant reduction in A during the dry period in both treatment areas (Figure 2A).A comparison of the species revealed higher A and g s values in X. sericea in the treatment area with plant litter removal during the dry period.
The chlorophyll a luorescence parameters showed no signiicant differences between the treatment areas for either X. sericea or S. guianensis (Figure 3).The parameters F o , F m , F v /F m , and F m /F o did not exhibit seasonal variation (Figures 3A,3B,3C,and 3D).In S. guianensis, NPQ was higher during the dry period in both treatment areas (Fig. 3F).When comparing the species, S. guianensis had a higher F m value in the litter removal treatment during the dry period (Figure 3B).were no significant differences between the treatment areas.This species also showed higher NPQ levels in both treatment areas during both the dry and rainy periods (Figure 3F).X. sericea exhibited higher qL in the treatment area with plant litter removal during the dry period (Figure 3E).
The photosynthetic pigment levels are presented in Table 1.There were no signiicant differences between the treatments (control and litter removal) for either species.Seasonal variations were observed with the exception of chlorophyll b.Chlorophyll a values were higher during the dry period, with the exception of S. guianensis in the treatment area with plant litter removal.Carotenoid content was higher for both species and treatment areas (control and litter removal) during the dry period, consequently reducing the total chlorophylls/ carotenoids ratio.The chlorophyll a/b ratio was higher during the dry period, except for S. guianensis in the control area.The highest level of total chlorophylls occurred in X. sericea during the dry period (treatment area with plant litter removal).When comparing the two species, only the level of carotenoids was higher in X. sericea in the treatment area with plant litter removal during the dry period.

Discussion
The removal of plant litter for 5 years did not affect photosynthetic parameters.The litter layer acts as a physical barrier, intercepting direct rainfall and solar radiation (BENKOBI et al., 1993;PONGE et al., 1993), reducing evaporation from the soil surface, and thus regulates soil microclimate (SAYER, 2006).Scalon et al. (2014) in a manipulation experiment (four years), showed that the removal of litter caused a decrease in g s and E in plants of the Cerrado, indicating that savanna woody plants responded promptly to litter manipulation by adjusting leaf water loss, which suggests that in the short term, changes in the amount of litter in Cerrado ecosystems can affect the soil water availability to the plant community.However, the authors emphasize that changes in some physiological traits may be dependent on a time-response to treatment, so 4 years may not be suficient time to allow us to observe a signiicant response, as in the present study.The litter layer is also an important source of nutrients for tropical ecosystems (VITOUSEK, 1984).However, as for the photosynthetic parameters, new and mature leaves of X. sericea did not show alterations in the concentrations of C, N, K, P, and Mg resulting from the removal of plant litter 4 years after the initiation of this manipulation in the União Biological Reserve (CAMARA, 2012).
According to Sayer (2006), some long-term studies done on plant litter manipulation found reduced concentrations of P, K, and Mg in soil only after 12 and 15 years in temperate regions.Although it was expected that the removal of plant litter would promote the rapid reduction of nutrient concentrations in tropical forests in their studies of a mature tropical forest and a secondary forest in Puerto Rico over two years, Wood et al. ( 2009) also found no forest or nutrient cycling productivity response as result of such manipulation.Sayer and Tanner (2010) obtained similar indings to those of Wood et al. ( 2009) in another study on plant litter manipulation on a large scale in a tropical lowland forest in Panama conducted over 5 years.The authors suggest that the experimentation period may not have been long enough to inluence the growth of the plants (127 species), and that a longer study period may be required.
Signiicant seasonal variation in the photosynthetic parameters was observed.Under conditions of low water availability during dry period, the stomatal closing reduced transpiration and reduced internal concentrations of CO 2 as well as higher IWUE in the leaves of both tree species.Under conditions of low water availability, stomatal opening is one of the irst plant processes to be affected, causing a reduction in gas exchanges (NILSEN;ORCUTT, 1996;BUCKLEY et al., 1999).
Although both species showed signiicant seasonal  et al., 2010).In the present study, a comparison of the species revealed that in the treatment area with plant litter removal, X. sericea plants showed higher A values in the dry period.
In some cases a reduction in A may be attributed to effects triggered by photochemical (supply of ATP and NADPH to the Calvin-Benson cycle) and/ or biochemical problems (rubisco concentration and enzyme activity; regeneration of RuBP by the Calvin-Benson cycle) (REIS; CAMPOSTRINI, 2008).In the present study, lower qL values in S. guianensis were observed when compared with X. sericea in the treatment area with plant litter removal, indicating that X. sericea showed a higher fraction of oxidized PSII based on the "lake model" (KRAMER et al., 2004).Nevertheless, the values of F o and F m indicated lack of damage to the center of the reaction of PSII (P 680 ) (MAXWELL; JOHNSON, 2000;ROSENQVIST et al., 1991).
According to Björkman and Demmig (1987), the F v /F m ratio in healthy plants should be around 0.83.F v /F m ratio relects the potential quantum eficiency of PSII and is used as a sensitive indicator of plant photosynthetic performance (KRAUSE;WEISS, 1991;GONÇALVES et al., 2001).Thus, we assume that during the periods analyzed, the plants studied were not subjected to stress conditions that would compromise their photosynthetic performance.This result was corroborated by the F m /F o values, which showed no signiicant seasonal variation.
The decline in photosynthetic assimilation in S. guianensis was accompanied by a signiicant increase in NPQ and carotenoid levels as well as a reduction in the total chlorophyll/carotenoids ratio.NPQ is considered one of the principal mechanisms utilized by plants to mitigate or to prevent the damage caused by excess light energy reaching the photosynthetic apparatus (MAXWELL; JOHNSON, 2000).According to Demmig et al. (1987), carotenoids also aid in the dissipation of excess non-destructive light energy, preventing damage to the PSII.In a similar way, Faria et al. (1996;1998) demonstrated the existence of a high degree of coordination between stomatal behavior and photoprotective mechanisms for the species Quercus ilex and Q. suber.
Higher levels of chlorophyll a and the chlorophyll a/b ratio were observed during the dry period.Similar results were reported for Byrsonima sericea under ield conditions in the União Biological Reserve (SILVA et al., 2010).However, no change or reduction in chlorophyll Ecophysiology of tropical species in an eucalypt plantation content of plants under drought stress has been observed in different species, and its intensity depends on stress rate and duration (SCHELMMER et al., 2005;FOTOVAT et al., 2007;ARJENAKI et al., 2012).
We can conclude that the removal of plant litter did not affect gas exchange, chlorophyll a luorescence, and the content of photosynthetic pigments.However, we found that X. sericea was photosynthetically more eficient than S. guianensis during the dry period, but neither species was subjected to water-related stress.

FIGURE 1 :
FIGURE 1: Precipitation and temperature between October 2009 and September 2010 for the weather station at União Biological Reserve, Brazil.Δ minimum temperature, ▲ maximum temperature (Source: LAGE-PINTO et al., 2012).

FIGURE 2 :
FIGURE 2: Gas exchanges (mean ± standard error; n = 18) of X. sericea and S. guianensis in the control and plant litter removal treatment areas during the rainy period (■) and dry period (□), in the União Biological Reserve, RJ, Brazil.(A) net photosynthetic rate (A), (B) intercellular CO 2 concentration (C i ), (C) stomatal conductance (g s ), (D) transpiration (E), and (E) intrinsic water use eficiency (IWUE).Lower-case letters compare periods for the same species within each treatment area.Different letters indicate signiicant differences (p ≤ 0.05).Asterisk (*) indicates signiicant differences (p ≤ 0.05) between species within the same treatment area and period.There were no signiicant differences between the treatment areas.

FIGURE 3 :
FIGURE 3: Parameters of chlorophyll a luorescence (mean ± standard error; n = 18) of X. sericea and S. guianensis in the control and plant litter removal treatment areas during the rainy period (■) and dry period (□), in the União Biological Reserve, RJ, Brazil.(A) F o , minimum luorescence; (B) F m , maximum luorescence; (C) F v /F m , maximum quantum yield of FSII; (D) F m / F o , ratio of luorescence yields for open and closed states; (E) qL, photochemical quenching (lake model), and (F) NPQ, nonphotochemical quenching coeficient.Lower-case letters compare periods for the same species within each treatment area.Different letters indicate signiicant differences (p ≤ 0.05).Asterisk (*) indicates signiicant differences (p ≤ 0.05) between species within the same treatment area and period.There were no signiicant differences between the treatment areas.

TABLE 1 :
Level of photosynthetic pigments (nmol mm −2 ) in leaves (mean ± standard error; n = 18) of X. sericea and S. guianensis in the control and plant litter removal treatment areas during the rainy and dry periods in the União Biological Reserve, RJ, Brazil.Chl, Chlorophyll; C x +c , carotenoids.Lower-case letters compare rainy and dry period for the same species within each treatment area.Different letters indicate signiicant differences (p ≤ 0.05).Asterisk (*) indicates signiicant differences (p ≤ 0.05) between species within the same treatment area and period.There were no signiicant differences between treatment areas.