*3.4. Relationship between* δ*13CR and Environmental Factors*

The correlation analyses showed that the δ13CR in the trunks of both *P. koraiensis* and *F. mandshurica* had a significant negative correlation with air temperature, soil temperature, and soil moisture (*p* < 0.01; Figure 6 and Table 1). Soil δ13CR only showed a significant positive correlation with global radiation (*r* = 0.312, *p* < 0.05; Figure 6 and Table 1). No significant correlation was found between leaf δ13CR and any environmental factors (Figure 6 and Table 1).

We conducted a correlation analysis between δ13Ceco and environmental factors with a time window from zero to 10 days. It was found that δ13Ceco was negatively correlated with air temperature (*p* < 0.05; Table 2) measured on the same day as δ13Ceco. And δ13Ceco was negatively correlated with VPD (*p* < 0.05; Table 2) and positively correlated with soil moisture (*p* < 0.01; Table 2) with a lag time of 10 days. Further, δ13Ceco was negatively correlated with global radiation and positively correlated with soil temperature, but this correlation was not statistically significant (Table 2).

**Figure 6.** Relationship between the δ13CR of different ecosystem organic pools and environmental factors. The data shown are the δ13CR of each measurement and the environmental factors of the corresponding half hour when the δ13CR was measured. VPD, vapor pressure deficit; *Pk*, Korean pine (*P. koraiensis*); *Fm*, Manchurian ash (*F. mandshurica*); *Am*, Mono maple (*A. mono*); *Qm*, Mongolian oak (*Q. mongolica*).

**Table 1.** Pearson correlation coefficients for the δ13CR from the C pools with environmental factors in the growing season. The δ13CR of each measurement and the corresponding half-hour meteorological data were used to conduct this analysis.


Note: \* *p* < 0.05, \*\* *p* < 0.01.

**Table 2.** Pearson correlation coefficients for the δ13Ceco with environmental factors in the growing season. δ13Ceco and daily average meteorological data were used to conduct this analysis. The lag time in days and the Pearson's correlation coefficient (r) are presented, and only the correlation that provided the best *r* value is presented.


Note: \* *p* < 0.05, \*\* *p* < 0.01.

#### **4. Discussion**

#### *4.1. Variations in Carbon Isotope Discrimination from Assimilation to Respiration*

Photosynthetic C isotope discrimination and δ13Cair both influenced leaf δ13C. In this study, the influence of photosynthetic discrimination on leaf δ13C was greater in the growing season most of the time. For example, from May to July, although δ13Cair showed a moderate increase, both Δleaf and Δcanopy showed a strong increase, which directly led to a continuous decrease in leaf δ13C during this period (Figure 3). This increase in Δleaf and Δcanopy may have resulted from an increase in stomatal conductance under the gradually warmer and wetter weather conditions from May to July (Figure 2). However, the influence of photosynthetic discrimination may have become weak for determining the leaf δ13C in August, at which point we also found that leaf δ13C reached its minimum, and Δleaf and Δcanopy were not the highest (Figure 3). On the other hand, δ13Cair influenced leaf δ13C more profoundly in terms of height. Since 13C depleted CO2 relative to the atmospheric CO2 respired by the soil and understorey and due to the reduction in mixing between the air inside the forest and the atmosphere with a decrease in height, canopy δ13Cair was usually lower, as it was closer to the ground [38,39], which resulted in a similar spatial pattern between leaf δ13C and δ13Cair (Figure 5).

The seasonal pattern of photosynthetic C isotope discrimination was found to be species-specific. Compared with deciduous broadleaf species, the coniferous species (*P. koraiensis*) was found to have the highest Δleaf in the growing season (Figure 3), which is consistent with a previous study conducted at the same site as our study [40]. Low photosynthetic activity and higher stomatal conductance could result in a higher ci/ca and, consequently, higher Δleaf [41]. Therefore, *P. koraiensis* in this temperate mixed forest may have a lower photosynthetic rate and higher stomatal conductance compared to deciduous species. Moreover, δ13C in the needles of *P. koraiensis* showed smooth depletion during the growing season, resulting in less varied Δleaf over time compared to the deciduous broadleaf species (Figure 3). This difference is most likely due to the large proportion of slow-turnover compounds (e.g., starch and pinitol) in needles compared to broadleaves. These compounds generally have more stable seasonal δ13C variation than other carbohydrates [11,42,43] and can strongly dampen the overall δ13C signals of needle bulk organic matter [43,44].

Our study also found apparent post-photosynthetic C isotope discrimination among the C pools from leaf to trunk and root (Figure 5). More importantly, our results show that the leaf (leaves of *P. koraiensis* and *A. mono*) and trunk (trunks of *P. koraiensis* and *F. mandshurica*) respired CO2 that was 13C-enriched in comparison to the leaf and trunk organic matter of the domain species (Figure 5). Due to the long duration of a single measurement (see Section 2.4), it was unfeasible to establish more chambers to cover more species and at different heights or to study more replicates. Despite this weakness in the δ13CR measurements, the results obtained by our study are more abundant than those in many previous studies [20,45,46] and indicate the effects of post-photosynthetic C isotope discrimination in the process of dark respiration. In the process of dark respiration, 13C-enriched C-3 and C-4 positions in glucose are preferably used to produce pyruvate during glycolysis; then, pyruvate is decarboxylated by pyruvate dehydrogenase (PDH) to release 13C-enriched CO2. A greater contribution of CO2 decarboxylated in the PDH will result in more positive δ13CR [47].

Soil δ13CR was more positive than δ13C in the potential respiratory substrates, including roots, litter, and soil on the surface; however, it was similar to δ13C in the deeper soil (Figure 5). Soil respiration is mainly performed by autotrophic components (roots and rhizosphere microbes) and heterotrophic components (the decomposition of litter and soil organic matter). Among the autotrophic components, root respiration contributes significantly to soil respiration with a generally more negative δ13CR signal compared to δ13C in root organic matter, as reviewed in [4,46,48]. Along with the relatively negative root δ13CR, if we further assume that litter δ13CR was mildly more positive than δ13C in litter organic matter during the process of decomposition, as shown in previous studies [49,50], more positive δ13CR signals would still be needed from other belowground processes to produce the exact soil δ13CR values in our forest, considering that litter respiration generally contributes less to soil respiration. Many studies have shown that δ13C in microbial biomass is more positive (1–2‰) relative to that in total soil C [4,51,52], and that microbial CO2 is also more 13C-enriched compared to soil organic matter [53]. Furthermore, the δ13C in soil CO2 seems to behave similarly to the typical patterns of soil δ13C isotopic enrichment with soil depth. For example, Goffin et al. [54] found that soil air δ13C in the deeper layer (10–20 cm) was more positive than that on the surface (0–10 cm) in a Scots pine stand. Using incubated soil, Formánek and Ambus [55] also found more positive δ13C in the CO2 respired from the deeper layer (15–38 cm) than that from the surface (3–10 cm). Thus, the soil δ13CR values observed in this study may have resulted from mixing between the δ13C signal from autotrophic respiration and a relatively large portion of the δ13C signal from microbe decomposition of soil organic matter, especially soil organic matter in the deeper layers, which indicates that the soil CO2 flux in our forest was mainly from heterotrophic components.
