Nitrogen input by bamboos in neotropical forest: a new perspective

Study area

The study was conducted in an Atlantic Forest region in northeastern São Paulo state, Brazil, in the Serra do Mar State Park (PESM in Portuguese) (Fig. 1). We selected 100 sample units (100 m² each) within previously established permanent plots (Joly et al., 2012). The physiognomy is pristine montane Atlantic Forest (1,000 m a.s.l.), with a humid subtropical climate (Cfa and Cfb), average annual temperature of 21 °C, average annual rainfall of 2,180 mm, and no dry season (Salemi et al., 2013). A dense fog covers the region almost daily, especially in winter. The soil order is Inceptisol (United States Department of Agriculture taxonomy), with low pH (≈3.8) and fertility, and high aluminum saturation (Martins et al., 2015). Both aboveground biomass (283.2 Mg ha⁻¹) (Alves et al., 2010) and floristic diversity (∼200 tree species ha⁻¹) (Padgurschi et al., 2011) are high (Joly, Metzger & Tabarelli, 2014). The most abundant families are Arecaceae, Myrtaceae, Lauraceae and Sapotaceae (Padgurschi et al., 2011).

Bamboo species: density, leaf area and litterfall

Merostachys neesii Rupr. (Poaceae: Bambusoideae), a native species of the Brazilian Atlantic Forest (Fig. 2), prefers humid, high-altitude environments (Judziewicz et al., 1999). All the clumps and live culms in the 100-sample units were counted (culm density) and culm density was compared against the highest density species in the area (Euterpe edulis Mart. Arecaceae—Padgurschi et al., 2011).

Habitat availability (bamboo leaf area) was estimated in order to determine N input by free-living diazotrophs in the phyllosphere. We calculated the total bamboo leaf area (LA_t) based on (i) culm density; (ii) leaf biomass per culm (L_b); and (iii) specific leaf area (SLA). L_b was previously determined by MCG Padgurschi, TS Reis, LF Alves, SA Vieira, CA Joly (2018, unpublished data) via destructive harvesting of 20 healthy culms around the study area (L_b = 506 g; 95% bootstrap, confidence interval: 316.2 and 701.2 were the lower/upper limits, respectively). For SLA, we randomly chose 50 bamboo leaves in the field, dried at 65 °C until constant weight, weighed to obtain the dry weight, and the leaf area was calculated using an LI-3100 area meter (LI-COR, Lincoln, Nebraska, USA). The leaf dry weight and leaf area (n = 50) were then used to calculate SLA. Leaf area per culm (LA_c) was determined as follows: (1)${\displaystyle L{A}_c=L_b\ast SLA}$ and total bamboo leaf area (LA_t) (m² ha⁻¹) by: (2)${\displaystyle L{A}_t=\frac{L{A}_c\text{*n culms}}{10000}}$ where “n culms” is the culm density within the sample units.

Among the 100 sample units, we randomly selected 40 to install circular litter traps (0.22 m² each). The traps were made of malleable plastic pipes with nylon mesh (2 mm) and supported by PVC pipes about 1 m above the ground. The content of the traps was collected twice a month over a year, from April 2014 to April 2015, sufficient time to capture this variable (Malhi, Doughty & Galbraith, 2011). For each collection, the bamboo leaves were separated, dried (at 65 °C until constant weight) and weighed to obtain dry weight. We calculated the production of bamboo litterfall in accordance with Sylvestre & Rosa (2002): (3)${\displaystyle \mathrm{LP}=\frac{\left(\frac{\sum \mathrm{MA}\ast 10,000}{\mathrm{CA}}\right)}{1000}}$ where LP = annual litter production (kg ha⁻¹y⁻¹); MA = average monthly litter production (kg ha⁻¹); CA = litter collector area (m²). For N chemical analysis of the bamboo leaves, we randomly selected three samples for each season (summer, fall, winter, spring) and ground them to obtain a compound sample per season (results are expressed in kg N ha⁻¹). The analysis was performed at the Soil and Plant Laboratory (LAGRO), in São Paulo, Brazil, using the Kjeldahl method of N determination. The study was performed with permits COTEC/IF 010.323/2013, 002.766/2013 and 010.631/2013 and IBAMA/SISBIO #33217.

Estimating N input by free-living N fixers in the M. neesii phyllosphere

To estimate N input by free-living diazotrophs on bamboo leaves, we used BNF rates previously recorded in the M. neesii phyllosphere at the same site studied here (Gómez, 2012). Gómez (2012) estimated BNF rates by acetylene reduction activity (ARA) based on a theoretical conversion ratio of 3:1 (reduction of three acetylene moles for each N mole fixed) (Hardy et al., 1968). The BNF rate in the bamboo phyllosphere was 64.25 ng N cm⁻² h⁻¹in winter and 34.78 ng N cm⁻² h⁻¹ in summer and, given the significant difference between these two values (Gómez, 2012), calculations for each season were performed separately.

Since light and temperature are important variables that affect microbial activity (Bentley, 1987; Reed, Cleveland & Townsend, 2011), we also considered the differences in hours of light during seasons. As such, based on available photosynthetically active radiation (PAR) data provided by the Climate and Biosphere Laboratory/Dept. of Atmospheric Sciences/University of São Paulo, bootstrapping (4,000 resamplings) was carried out to obtain the median and lower/upper limits of PAR (Table 1). We used the number of hours around the PAR median added to the lower/upper limits (828 ± 70 µmol m⁻² s⁻¹ in summer; 711.24 ± 55 µmol m⁻² s⁻¹ in winter; 95% confidence intervals) (Table 1). Finally, N fixing potential was estimated (N_f expressed in kg N ha⁻¹y⁻¹) as follows: (4)${\displaystyle N_f=\frac{\left(BNF\ast L{A}_t\right)\ast H_l}{10^{12}}}$ where H_l is the hours of light in summer or winter (Table 1).

N cycling

To contextualize the estimated N input mediated by M. neesii, data on the N cycling in the Atlantic Forest were obtained from the literature. The two dominant N input pathways (Hedin et al., 2009) considered were symbiotic BNF (Manarin, 2012) and total atmospheric N deposition (Groppo, 2010), in addition to the free-living N fixers on bamboo leaves (this study).

In terms of N required by the system (demand), we used litterfall to predict net primary productivity (NPP). The NPP fraction allocated to leaves influences litterfall rates, making it a good predictor of productivity in neotropical forests when the main components of NPP cannot be measured (Malhi, Doughty & Galbraith, 2011). Based on this principle, we used the literature data on ecosystem litter production (5.5 Mg ha⁻¹ y⁻¹—Sousa Neto et al., 2011) and the N content of the litter (1.72%—Vieira et al., 2011), as well as bamboo litter with its respective nitrogen concentration (see the “Bamboo species: density, leaf area and litterfall” section for details). The N content of litter is equivalent to the minimum amount required for tree and bamboo growth, since plants reallocate nutrients before leaf abscission, meaning litter exhibits lower N levels when compared to live leaves (Chapin III et al., 1987; Tripathi et al., 2006). The annual production of fine roots (<2 mm) was considered representative of demand. These roots represent at least twice as much carbon and nitrogen stock as that found aboveground in the AF (Vieira et al., 2011). Fine root production of 10 Mg ha⁻¹ y⁻¹ (Silva, 2015) and N content of 1.3% (Sousa Neto et al., 2011) were used.

Finally, riverine transport and N₂O and NO losses via soil emissions were included as outputs (Groppo, 2010; Sousa Neto et al., 2011; Ghehi et al., 2013). The NO emission we presented here is based on models developed for a tropical highland forest (Ghehi et al., 2013) similar to the study area, as follows: (i) pristine montane forest (1,000 m a.s.l.); (ii) 2,000 mm y⁻¹ of rainfall; (iii) presence of bamboo; (iv) pH 3.8 (Ghehi et al., 2013; Martins et al., 2015). All analyses and graph were performed using R environment (R Core Team, 2014).