Ideas and perspectives: patterns of soil CO 2 , CH 4 , and N 2 O ﬂuxes along an altitudinal gradient – a pilot study from an Ecuadorian neotropical montane forest

Abstract. Tropical forest soils are an important source and sink of greenhouse gases
(GHGs), with tropical montane forests, in particular, having been poorly studied. The
understanding of this ecosystem function is of vital importance for future
climate change research. In this study, we explored soil fluxes
of carbon dioxide (CO2), methane (CH4), and nitrous oxide
(N2O) in four tropical forest sites located on the western flanks of
the Andes in northern Ecuador. The measurements were carried out during the
dry season from August to September 2018 and along an altitudinal gradient
from 400 to 3010 m a.s.l. (above sea level). During this short-term campaign, our measurements
showed (1) an unusual but marked increase in CO2 emissions at high
altitude, possibly linked to changes in soil pH and/or root biomass, (2) a
consistent atmospheric CH4 sink over all altitudes with high temporal
and spatial variability, and (3) a transition from a net N2O source to
sink along the altitudinal gradient. Our results provide arguments and
insights for future and more detailed studies on tropical montane forests.
Furthermore, they stress the relevance of using altitudinal transects as a
biogeochemical open-air laboratory with a steep in situ environmental gradient
over a limited spatial distance. Although short-term studies of temporal
variations can improve our understanding of the mechanisms behind the
production and consumption of soil GHGs, the inclusion of more rigorous
sampling for forest management events, forest rotation cycles, soil type,
hydrological conditions and drainage status, ground vegetation composition
and cover, soil microclimate, and temporal (seasonality) and spatial
(topographic positions) variability is needed in order to obtain more
reliable estimates of the CO2, CH4, and N2O source/sink
strength of tropical montane forests.


Organization for Standardization (ISO 11277:2009), soil texture was determined. The classification of the soil was made according to the United States Department of Agriculture (USDA, 2017); and the soil class was determined based on the classification of FAO and UNESCO: World Reference Base for Soil Resources (WRB) (FAO, 2007). Daily measurements of soil moisture, expressed as water-filled pore space (WFPS), were taken per site at 5 and 20 cm depth 45 using soil moisture sensors Meter Environment,Pullman,Washington,USA) and data loggers (ProCheck, Meter Environment, Pullman, Washington, USA). Finally, soil temperature was determined daily for each measurement cycle and per chamber, by means of a thermometer inserted at 5 cm depth and approximately 10 cm from each chamber.

Soil-atmosphere exchange
Each day, the chambers were closed for a period of 1 h. Samples of 20 mL were taken with disposable syringes from the 50 headspace air every 20 minutes: T1 = 0, T2 = 20, T3 = 40 and T4 = 60 min; T1 or time-zero indicates the sample taken immediately after the chamber was closed. Before each sample collection, the syringe was flushed twice with the air of the chamber to mix the chamber headspace and to avoid any possible stratification of gases.
The 20 mL samples were injected in pre-evacuated 12 mL exetainer vials (over-pressurized), and once the sampling campaign 55 was over, the samples were sent to Belgium (Ghent University) for gas chromatography analysis. For CH4 and CO2 analysis, a gas chromatograph (Finnigan Trace GC Ultra; Thermo Electron Corporation, Milan, Italy) equipped with a flame ionization detector (FID) and a thermal conductivity detector (TCD) was used, respectively. For N2O, another gas chromatograph equipped with an electron capture detector (ECD) (Shimadzu GC-14B; Shimadzu Corporation, Tokyo, Japan) was used.

Data analysis 60
The fluxes for each gas (CO2, CH4 and N2O) were calculated by means of linear regressions using the four consecutive measurements of each measurement cycle. The slope of the regressions represented the flux. Thus, following the ideal gas law, and considering the headspace volume of the chamber and the chamber area, the net gas flux was calculated by Eq. (2) (Collier et al., 2014;Dalal et al., 2008;Kutzbach et al., 2007): where Fc corresponds to the net gas flux (CO2, CH4 or N2O), ∆c/∆t is the rate of change of the gas concentration within the chamber or the slope of the regression line [ppm min -1 or µL L -1 min -1 ], P/RT corresponds to the ideal gas law used to convert concentration from volumetric to mass fraction at local temperature and pressure, R = gas law constant (0.08206 L atm mol -1 k -1 ); MW is the molecular weight of the gas (CO2-C and CH4-C: 12.01 g mol -1 , N2O-N: 14.01 g mol -1 ), Vch is the headspace volume of the chamber, and Ach the area of the chamber. The goodness-of-fit was evaluated for every linear regression using 70 the adjusted coefficient of determination (R 2 ), and time series (concentration vs time) with R 2 < 0.65 were excluded from further analysis.
All soils along the altitudinal gradient are Andosols and the soil texture was classified (USDA) between loam and sandy loam at all sites (WRB; Table 2). All sites had a relatively acidic soil; pHwater ranged from strong to medium acidic (4.6 -5.7), with an increase in acidity with depth, except at P_3010 ( Table 2). The most acidic soil was found at S_400 at 5 cm, although not significantly different from M_1100 and C_2200; whereas the least acidic one at P_3010 at 5 cm depth, and only significantly different from M_1100. Except for P_3010, NO3-N concentrations were 2-4 times higher at 5 cm compared to 20 cm depth; 80 the highest variability was observed at S_400, and in comparison to the other sites, P_3010 seems to be depleted in NO3-N at both depths (0.8 -3.6 µg g -1 ). In contrast, the highest concentration of NH4-N was obtained at P_3010 at 20 cm, followed by S_400 at 5 cm. However, at all sites, NH4-N concentrations at 5 cm were not significantly different from each other. Such as NO3-N, NH4-N also decreased with depth, except at P_3010 where the increase at 20 cm with respect to 5 cm was almost doubling. Higher N contents were measured at 5 cm compared to 20 cm depth at all sites; and S_400 exhibited the highest 85 content at both depths, 1.3-1.4 times higher than any other N percentage at the same depth, and even 4 times higher than any other N percentage at 20 cm depth. The C content showed a general decrease with depth at all sites, with the highest percentage at S_400 at 5 cm, and the lowest one at M_1100 at 20 cm. Higher δ 15 N signatures were obtained at 20 cm compared to 5 cm depth; at S_400 the soil was most enriched in 15 N, and P_3010 showed the most depleted one.

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Soil temperature decreased with altitude with a gradient of -4.2 °C per 1000 m, with no statistical difference between months ( Fig. S2). WFPS increased significantly with depth at all sites during both months (Fig. S3), except at C_2200 in September.

Greenhouse gas fluxes 95
In general, all sites were sources of CO2 (Fig. 1a, Table 1). Except for P_3010, mean CO2 emissions were higher in September compared to August, but due to the high variability in the measurements, there was no significant difference between months at M_1100, C_2200 and P_3010 (P > 0.05). The lowest and highest emissions were observed at C_2200 (August and September) and P_3010 (August), respectively. All mean CH4 fluxes were negative, indicating a net flux from the atmosphere to the soil (Fig. 1b, Table 1). Although the mean CH4 fluxes (except for P_3010) were higher in September compared to 100 August, there was no significant difference (P > 0.05) between months at any site. Finally, the mean N2O fluxes showed a general negative trend with increasing altitude (Fig. 1c). A marked net N2O consumption was observed at the sites located at 2200 and 3010 m a.s.l.; however, there was no significant difference (P > 0.05) in any plot between months. The highest consumption was observed in September at C_2200, followed by P_3010 in August, while the highest emission was in September at M_1100 (Table 1). 105 Although only monthly average fluxes were discussed, the large variability observed with most of the gas fluxes (Table 1 and 4 Average of the annual rainfall measured at the experimental site during 4 years. 5 Cumulative N2O fluxes from the 11/05/2010 to the 09/05/2011 6 Values for undistrubed "native" soil. This study evaluated the climate dependence of heterotrophic soil respiration from a soil-translocation experiment. As a matter of comparison 135 with the others studies, only control cores are depicted, i.e. soil cores re-installed at the same site. Table S2. Characteristics of the study areas Río Silanche (400 m a.s.l.; S_400), Milpe (1100 m a.s.l.; M_1100), El Cedral 140 (2200 m a.s.l.; C_2200) and Peribuela (3010 m a.s.l.; P_3010), including mean annual precipitations (MAP) and mean annual temperatures (MAT) extracted from the Worldclim data set, using average monthly data from 1970-2000 with a spatial resolution of ~1 km 2 (Fick and R.J. Hijmans, 2017). Forest classification has been done based on the system used by the country (FAO, 2017;Ministerio del Ambiente, 2015). Note: the coordinates were taken at the center of the plots.