*2.4. Carbon Isotope Composition of CO2 Respired from the Leaves, Trunks, Soil, and Ecosystem*

Five ports of the profile control system along with the respiration chambers were used to build a closed-circuit system under non-steady-state conditions [21,32] to conduct measurements of the δ13C in the leaf-, trunk-, and soil-respired CO2 (δ13CR) (Figure 1a). The outlet of each chamber was directly connected to the rotation valve in the profile control system and then connected to the inlet of the WS-CRDS analyzer. The outlet of the WS-CRDS analyzer, which is the outlet of the Picarro external pump, was divided into five sub-outlets by a 5-way air distribution manifold connected to the inlet of each chamber. All tubes were 1/8-inch Teflon tubes, thereby reducing the system volume and response time.

The leaf chambers were made from cylindrical PET (polyethylene terephthalate) jars (10 cm in diameter and 20 cm in height) (Figure 1b,c). The lid of each jar was fixed on the branch by letting the branch pass through the center of the lid. Mastic tape and silicone sealant were used to fill the space between the branch and the lid to make the leaf chamber airtight. The inlet and the outlet were on the sides and the bottoms of the jar bodies. No fans were installed inside the jars because of limited space.

The trunk chambers were made from cylindrical PP (polypropylene) airtight containers (18 cm in diameter and 9.5 cm in height) (Figure 1b,c). The bottoms of each container were removed, and the rest of the body was fixed onto the trunk using silicone sealant after gently sanding the bark. An inlet and an outlet were located on the lid of the container. A mini fan was installed inside the lid for air mixing.

The soil chamber was made from the same material and of the same size as the trunk chambers (Figure 1b,c). The body of the soil chamber was fixed on a soil collar (16 cm in diameter and 10 cm in height) using hot-melt adhesive. The soil collar was inserted into the soil to a depth of 5 cm. Here, only one soil chamber was used due to the limited number of sampling ports. The leaf chambers and the soil chamber were permanently covered by aluminum foil tape to isolate sunlight and maintain a steady temperature.

Two leaf chambers were installed on a branch of a *P. koraiensis* and an *A. mono*. The branches were mature and healthy with a diameter of about 1.5 cm and located at about 5 m in height on the sunny side of the trees. Two trunk chambers were installed on the sunny side of the trunks on a *P. koraiensis* and a *F. mandshurica* at breast height (1.3 m). The selected trees and the soil collar were about 5 m apart from each other, thereby sharing similar meteorological and environmental conditions. The chambers were installed one month before the first sampling. Each of the chambers included a silicon sealing ring on the inside of the lid to ensure airtightness. The airtightness of the respiration chamber was tested by connecting a CO2/H2O analyzer (LI-850, LI-COR, Lincoln, NE, USA) to a closed chamber (screwing the body back on the lid for leaf chambers or locking the lid with clamps on the body for trunk and soil chambers). If there was no obvious change in the measured values of CO2 concentration in the chamber when blowing along the joints of the chamber, then the sealing performance was considered good.

The δ13CR measurements were conducted from early May to late September of 2019. To begin measuring, the chamber was first closed after selecting an inlet and an outlet corresponding to the chamber by manually operating the rotation valve and the air distribution manifold. The measurement continued until the internal CO2 concentration increased by about 200 ppm (about 20–40 min for the leaf and trunk chambers and about 5–10 min for the soil chamber), as suggested in [21], and then we switched to the next chamber. The chambers were closed only during measuring. We periodically closed the chambers in the following sequence: the *P. koraiensis* leaf chamber (L1), the *A. mono* leaf chamber (L2), the *P. koraiensis* trunk chamber (T1), the *F. mandshurica* trunk chamber (T2), and the soil chamber (S1). Four rounds of measurements were conducted from around 7:00 to 18:00 each day. The δ13CR of each measurement was estimated based on the widely used Keeling plot approach [22,23]. The daily δ13CR of the leaf, trunk, and soil was averaged from the four measurements each day.

The δ13Ceco was determined by the Keeling plot approach [22,23] using the nighttime (21:00–03:00) CO2 and δ13Cair profile measurements. Considering that small a CO2 range would cause uncertainties in fitting the Keeling plot [24], we set a restriction that only nights with a CO2 range greater than 60 ppm were used to conduct the regression, and the ordinary least squares regression (OLS, model I) was used to obtain the intercept of regression, as suggested by Chen et al. [25].

#### *2.5. Sampling and Carbon Isotope Analysis of the Ecosystem Compartments*

Leaves of the five dominant species were collected in the mornings of sunny days once a month from May to September 2019 around the flux tower. Specifically, nine mature trees of each species were randomly selected on each sampling day; then, mature and healthy leaves were collected at three canopy heights (lower, middle, and upper). Since the trees are different in height and the canopy structure is different among species, the three canopy heights of each tree were determined relative to their canopy height range rather than using absolute heights. We collected bark and xylem at breast height and coarse and fine roots of the five species, as well as litters in the undecomposed layer (Oi) and decomposed layer (Oe + Oa) and the soil at four depths (0–5, 5–10, 10–20, and 20–40 cm) with nine replicates around the flux tower at the end of September 2019.

All samples were immediately transported to the lab and dried at 65 ◦C for 4 days to a constant weight and then ground with a ball mill (MM 400, Retsch, Haan, Germany). The C isotope compositions of nine sample replicates were determined separately using an elemental analyzer (Flash EA1112, Thermo Finnigan, Milan, Italy) coupled with a mass spectrometer (Finnigan MAT 253, Bremen, Germany). The overall precision of the <sup>δ</sup>13C measurement was <sup>&</sup>lt; <sup>±</sup>0.2‰. All <sup>δ</sup>13C values are reported on the VPDB (Vienna Pee Dee Belemnite) scale.

#### *2.6. Calculation of Photosynthetic Carbon Isotope Discrimination*

C isotope discrimination of the leaf (Δleaf) was calculated as

$$
\Delta\_{leaf} = \frac{\delta\_{air} - \delta\_{leaf}}{\delta\_{leaf} / 1000 + 1} \tag{1}
$$

where δair is the averaged C isotope composition of the canopy CO2 (‰) measured at 26 m within the canopy during the sampling day, and δleaf is the averaged C isotope composition of the leaves (‰) for all dominant species and canopy heights.

Canopy-scale photosynthetic discrimination (Δcanopy) was calculated using Farquhar's classical model (Δclassical) [1] and compared with Δleaf to provide robust evidence for variations in C isotope discrimination over time. Δclassical describes the fractionation in CO2 diffusion, carboxylation fractionation, and respiratory fractionation as

$$
\Delta\_{\text{classical}} = \overline{a} + (\mathbf{b} - \overline{a})\frac{c\_c}{c\_a} - f\frac{\Gamma^\*}{c\_a} - c\frac{R\_d}{kc\_a} \tag{2}
$$

where *a* is the overall diffusional fractionation for CO2 as calculated from the diffusional fractionation factors (fractionation across the boundary layer (2.9‰), fractionation across the stoma (4.4‰), and fractionation across the mesophyll, including dissolution (1.1‰ at 25 ◦C) and diffusion (0.7‰) in water) and the conductance factors of CO2 across the foliar boundary layer, stoma, and mesophyll during photosynthesis based on the big-leaf model according to Wehr and Saleska [33]; b is the Rubisco fractionation in C3 plants and assumed to be 27.5‰ [34]; f is the respiratory fractionation for photorespiration (11‰) [33]; e is the respiratory fractionation for daytime dark respiration (−5‰) [33]; Γ\* is the CO2 compensation point (μmol mol−1), which was calculated from the leaf temperature according to Brooks and Farquhar [35]; Rd is the daytime respiration rate (μmol m−<sup>2</sup> s−1), which is assumed to be 3 μmol m−<sup>2</sup> s−<sup>1</sup> [36]; k is the carboxylation efficiency (μmol m−<sup>2</sup> s<sup>−</sup>1), which is assumed to be 0.1 μmol m−<sup>2</sup> s−<sup>1</sup> [36]; cc is the CO2 concentration in the chloroplast (ppm); and ca is the CO2 concentration in the canopy air (ppm) measured at 26 m above the ground (see Section 2.3). The cc/ca was obtained by the numerical solution of the equation derived from the theory of isoflux-based isotopic flux partitioning (IFP) [19], which is expressed as

$$\frac{c\_d}{c\_d} = \frac{- (\delta\_a + b - \delta\_{NR} - 2\overline{\pi} + \frac{f\Gamma^\*}{c\_d} + \frac{eR\_d}{hc\_d}) \pm \sqrt{(\delta\_a + b - \delta\_{NR} - 2\overline{\pi} + \frac{f\Gamma^\*}{c\_d} + \frac{eR\_d}{hc\_d})^2 - 4(\overline{\pi} - b)(\delta\_{NR} + \overline{\pi} - \delta\_a - \frac{f\Gamma^\*}{c\_d} - \frac{eR\_d}{hc\_d} - \frac{i\alpha f \text{Im}\nu - \delta\_{NR}\text{NEE}}{\overline{\pi}^\* d})}{2(\overline{\pi} - b)}}{2(\overline{\pi} - b)}\tag{3}$$

where δ<sup>a</sup> is the δ13C of canopy CO2 measured at 26 m above the ground (‰) (see Section 2.3); δNR is δ13Ceco (see Section 2.4) but was substituted by the weekly smoothed value to capture the

seasonal variation; isoflux is the C isotopic flux calculated from the eddy covariance measurement (see Section 2.2), the canopy CO2 δ13C profile measurement (see Section 2.3), and the intercept of the daytime (7:00–17:00) Keeling plot according to [18,37]; and NEE is the net ecosystem exchange flux (μmol CO2 m−<sup>2</sup> s<sup>−</sup>1). Note that Δcanopy was calculated using data from the daytime and expressed as the daily average.

### *2.7. Statistical Analyses*

Student's *t*-test was used to assess the differences in mean δ13C (air, organic matter, and respiration) among sampling positions, C pools, or species. A one-way analysis of variance (ANOVA) was performed to assess the significance of the seasonal variations of δ13Cair, leaf δ13C, Δleaf, δ13CR, and δ13Ceco. To evaluate how the δ13CR of the leaf, trunk, and soil respond to environmental factors, we conducted correlation analyses and linear regression analyses using the δ13CR of each measurement and the environmental data of the corresponding timespans. Correlation analyses were also conducted to assess the environmental effects on δ13Ceco and the potential lagged responses of δ13Ceco to environmental factors using daily δ13Ceco and daily average meteorological data. The environmental factors include air temperature, vapor pressure deficit (VPD), global radiation, soil temperature, and soil moisture. These factors were taken into consideration because they are expected to influence C isotope discriminations in above- and belowground processes. The lagged correlation was tested with shifted time periods from zero to 10 days.

#### **3. Results**
