*3.1. Seasonal Variation of* δ*13Cair*

In the year 2019, the mean annual temperature was 5.4 ◦C and annual rainfall was 627 mm, making 2019 warmer and drier than the long-term average (see Section 2.1). During the growing season of 2019, the meteorological conditions showed significant seasonal fluctuations (Figure 2). The vapor pressure deficit (VPD) was higher in May and September, and air temperature was also higher during these periods, which resulted in remarkable soil water deficits. Soil temperature and air temperature reached their highest points at the end of July. In July and August, precipitation and soil moisture were the highest, especially at the end of July and in the middle of August, when the accumulated rainfall accounted for 15.7% and 19.6% of the annual total, respectively.

We found a significant seasonal variation in δ13Cair during the growing season (*p* < 0.01, ANOVA; Figure 3a). The average <sup>δ</sup>13Cair of the growing season was <sup>−</sup>9.25‰. At the beginning of the growing season, <sup>δ</sup>13Cair showed a slight decrease and then reached a maximum of about <sup>−</sup>8.5‰ in early July and then started to decrease until it reached a minimum of below −10‰ in the middle of August. In September, δ13Cair first showed an increase and then started to decrease in the middle of September. Moreover, δ13Cair showed remarkable day-to-day variation. The largest difference in δ13Cair between two consecutive days was up to about 2‰. Short-term rapid depletions of 13C in the forest occasionally occurred during the growing season, such as the relative depletion of 13C at the beginning of September. The difference of <sup>δ</sup>13Cair between the above-canopy (−9.13‰) and within-canopy (−9.18‰) was small but statistically significant (*p* < 0.01, *t*-test). δ13Cair within the canopy was significantly more positive (*<sup>p</sup>* <sup>&</sup>lt; 0.001, *<sup>t</sup>*-test) than the <sup>δ</sup>13Cair below the canopy (−9.44‰), and <sup>δ</sup>13Cair below the canopy was always the lowest throughout the growing season.

**Figure 2.** Seasonal variations in global radiation (**a**), vapor pressure deficit (VPD) (**b**), air and soil temperature (**c**), and soil moisture and precipitation (**d**) during the 2019 growing season. Global radiation, VPD, and air temperature are the averages of the seven heights in the forest. Soil temperature and soil moisture were measured at a 5 cm depth.

**Figure 3.** Seasonal variation in the δ13C of atmospheric CO2 (δ13Cair) (**a**), seasonal variation in the δ13C of leaf bulk organic matter (**b**), and C isotope discrimination at the leaf and canopy levels (**c**) during the growing season of 2019. δ13Cair values are given as daily averages. The δ13Cair values below the canopy were averaged from data collected at heights of 2.5 and 8 m; the δ13Cair values within the canopy were averaged by data collected at heights of 22, 26, and 32 m; the δ13Cair values above the canopy were averaged by data collected at heights of 50 and 60 m. The dashed line is the locally weighted scatterplot smoothing (LOESS) with span = 0.3. The two periods of missing data in June and August were caused by power failure. Leaf <sup>δ</sup>13C values (mean <sup>±</sup> SE, n <sup>=</sup> 9) were aggregated from three canopy heights (lower, middle, and upper). Δleaf was calculated from leaf δ13C and δ13Cair and separated by species; gray circles represent Δcanopy derived from Farqhar's Δclassical model. *Pk*, Korean pine (*P. koraiensis*); *Fm*, Manchurian ash (*F. mandshurica*); *Am*, Mono maple (*A. mono*); *Qm*, Mongolian oak (*Q. mongolica*); *Ta*, Tuan linden (*T. amurensis*). Error bars indicate standard deviation.

#### *3.2. Carbon Isotopic Compositions of Leaves and Photosynthetic Carbon Isotope Discrimination*

Considering the complexity of the mixed forest, we determined the leaf δ13C for all five dominant species and along the vertical position of their canopies. In terms of temporal variation, leaf δ13C in broadleaved species showed significant seasonal variations (*p* < 0.001, ANOVA) except for *A. mono* (*p* = 0.516, ANOVA), with a gradual decrease from May to August by a magnitude of 2‰, followed by an increase after August (Figure 3b). In contrast to broadleaves, the δ13C in the needles of *P. koraiensis* did not vary significantly over time (*p* = 0.275, ANOVA) and showed a slight decrease throughout the growing season, except for a relatively small increase in July (Figure 3b). δ13C in the needles of *P. koraiensis* (−29.79‰) was also the lowest among the species (Figure 3b). Since broadleaved trees are the main components of the mixed forest in this study, variation in the mean leaf δ13C values of the five dominant species followed the same seasonal patterns as the four broadleaved species. The temporal variation of leaf δ13C corresponded to that of δ13Cair, except in May, when δ13Cair was relatively low, while leaf δ13C was the highest compared to the rest of the growing season (Figure 3a,b), indicating that leaf δ13C depends not only on the C isotopic signal carried by CO2 in the air.

C isotope discrimination at both the leaf and canopy level is shown in Figure 3c. Δleaf was calculated directly from the leaf δ13C and δ13Cair of the corresponding sampling days (Equation (1)). Species-specific differences in Δleaf were found over the growing season. Except for *A. mono* (*p* = 0.084, ANOVA), Δleaf varied significantly (*p* < 0.05 for *P. koraiensis* and *p* < 0.001 for the other species, ANOVA) over time. On average, Δleaf showed an increasing trend from May (18.97‰) to July (20.70‰), while the Δleaf of both species showed a decrease in August compared to July and September (Figure 3c). This variation pattern of leaf level discrimination was strongly supported by the Δcanopy estimated by the classical Farquhar's model (Equation (2)), although Δcanopy was generally lower than Δleaf (Figure 3c). Δcanopy also showed a general increasing trend over time, with the lowest value observed in May. Since there are two periods of missing measurements, we can only deduce that the Δcanopy reached its maximum in early to middle July, followed by relatively lower values in the middle of August, according to the increasing trend in June and the decreasing trend at the beginning of August (Figure 3c).

## *3.3. Carbon Isotopic Compositions of Ecosystem Organic Pools and Respired CO2*

Seasonal variation in the δ13CR of different ecosystem pools (leaf, trunk, and soil), as well as δ13Ceco, is shown in Figure 4. Over the entire growing season, the δ13CR of leaves and trunks varied within a range of 3.2‰ and 4.5‰, respectively. A statistically significant general increase of δ13CR was found in the needles of *P. koraiensis* (*p* < 0.001, ANOVA), while no such phenomenon was found in the leaves of *A. mono* (*p* = 0.422, ANOVA; Figure 4a). The trunk δ13CR of both *P. koraiensis* and *F. mandshurica* varied significantly (*p* < 0.001, ANOVA) throughout the growing season. Trunk δ13CR showed a decreasing trend from May to August and a slight increase in September (Figure 4b). This variation trend was similar to the general seasonal pattern of leaf δ13C (Figures 3a and 4b). Soil δ13CR did not vary significantly over time (*p* = 0.084, ANOVA) and was also relatively stable (with a range of 2.3‰) compared to leaf and trunk δ13CR (Figure 4c). Furthermore, a decrease of δ13CR was found from May to June in leaf, trunk, and soil (Figure 4a–c).

<sup>δ</sup>13CR exhibited a slight depletion from leaves (−26.33‰) to trunks (−25.67‰) and soil (−25.48‰), as did <sup>δ</sup>13C in the corresponding organic matter (leaf: <sup>−</sup>28.68‰, trunk: <sup>−</sup>26.55‰, soil: <sup>−</sup>25.72‰; Figure 5). A distinct spatial pattern of leaf δ13C was found throughout the growing season (Figure 5). Similar to <sup>δ</sup>13Cair, leaf <sup>δ</sup>13C values increased significantly with an increase of height (−29.16‰, <sup>−</sup>28.73‰, and <sup>−</sup>28.16‰ from the lower to middle and upper canopy; *<sup>p</sup>* <sup>&</sup>lt; 0.001, ANOVA). Leaf <sup>δ</sup>13CR was generally more enriched compared to the δ13C in leaf organic matter but closer to the δ13C in the upper leaf (−28.16‰), which indicates that a substantial quantity of photosynthate from the upper canopy was used as respiratory substrate. Trunk δ13CR was also more enriched than the δ13C in trunk organic matter but showed a larger range of variation (Figure 5). Soil δ13CR was more enriched than the <sup>δ</sup>13C of organic matter on the surface (0–5 cm) soil (−26.59‰), in the roots (−28.02‰), and especially

in litters (−28.40‰). However, the soil <sup>δ</sup>13CR was similar (*<sup>p</sup>* <sup>=</sup> 0.527, *<sup>t</sup>*-test) to the <sup>δ</sup>13C of soil in deeper layers (5–40 cm) (−25.43‰; Figure 5).

Nights with a CO2 range lower than 60 ppm were excluded from δ13Ceco calculations because a small CO2 range could cause uncertainties in fitting the Keeling plot. After applying this exclusion, all of the values for R<sup>2</sup> in the regression were greater than 0.85 and yielded a sufficient quantity of reliable δ13Ceco data (Figure 4d). The variation in δ13Ceco was significant over the growing season (*p* < 0.001, ANOVA), with an average of <sup>−</sup>25.79‰. <sup>δ</sup>13Ceco steadily increased as the growing season progressed (Figure 4d), and the δ13Ceco values were within the range of the δ13CR in the leaves, trunks, and soil (Figure 5). Some short-term δ13Ceco variations were observed, such as two gradual decreases in May and July, which may be attributed to the gradual 13C depletion of CO2 in the air and respired from C pools during these periods (Figure 3; Figure 4).

**Figure 4.** Seasonal variations in the δ13CR of leaves (**a**), trunks (**b**), and soil (**c**), and in δ13Ceco (**d**). δ13CR and δ13Ceco were both derived using the Keeling plot method. The δ13CR of leaves, trunks, and soil were averaged from four measurements between 7:00 and 18:00, and the error bars indicate standard deviation. δ13Ceco was determined from the nighttime (21:00–03:00) CO2 and δ13Cair profiles, and the error bars indicate standard errors of the intercept of the Keeling plot. *Pk*, Korean pine (*P. koraiensis*); *Fm*, Manchurian ash (*F. mandshurica*); *Am*, Mono maple (*A. mono*); *Qm*, Mongolian oak (*Q. mongolica*).

**Figure 5.** A comparison of the C isotopic composition of ecosystem organic pools and respiratory fluxes. Diamonds represent the means. The box represents the median and the 25% upper/lower quartiles. The tails represent the 10% and 90% limits of the data. Leaf samples were taken once a month from May to September 2019. The trunk values are the average of the bark and xylem, the root values are the average of the coarse and fine roots, and the litter values are the average of the undecomposed layer (Oi) and the decomposed layer (Oe + Oa). These samples, along with the soil samples, were sampled at the end of September 2019. The leaf, trunk, and soil δ13CR were measured 15 times from May to September 2019. δ13Ceco was determined at a daily scale from May to September 2019.
