*4.3.* δ*13Ceco Reveals Short-Term Links between Aboveground and Belowground Processes*

The δ13Ceco calculated by the Keeling plot method using nighttime δ13C and CO2 profile data represents the integrated δ13C of the CO2 produced by all above- and belowground respiring components in the forest. The results showed that the average δ13Ceco in the growing season is between the average δ13CR of the trunk and the soil (Figure 5), which is consistent with the results reported by Wingate et al. [17].

From a short-term perspective, aboveground vegetation could determine the biogeochemical processes belowground through, for example, newly fixed photoassimilates transport to roots, which then contribute to soil respiration. This linkage between assimilates and respiration could be revealed by relating the δ13C of ecosystem respiration to environmental factors but with a time lag from hours to days [9,10,16,61]. For example, Fessenden and Ehleringer [59] found that soil moisture measured at a 40 cm depth was negatively correlated with δ13Ceco on the day of the measurements in an old-growth coniferous forest. Scartazza et al. [62] also found a negative relationship between soil moisture measured at 70–88 cm depth and δ13Ceco measured on the same day in a beech forest, whereas Mortazavi et al. [6] found that δ13Ceco was negatively correlated to soil moisture for 7 days before δ13Ceco measurements. The negative correlation between soil moisture and δ13Ceco was also found to be significant in a Douglas-fir forest but not in a ponderosa pine forest [63]. However, our results suggest that the correlation between soil moisture and δ13Ceco is positive but weak and that a lag of 10 days provides the best correlation (Table 2).

The VPD was found to be positively related to δ13Ceco after a lag of 3 days [63], 3–4 days [6], 4–5 days [14,61], or 5–10 days [16]. Again, our results show an opposite correlation pattern between VPD and δ13Ceco compared to previous studies, and a lag of 10 days gave the best (but still weak) correlation (Table 2). In previous studies, Bowling et al. [64] and Schaeffer et al. [65] found no correlation between δ13Ceco and soil moisture or VPD. We also found that δ13Ceco has no significant correlation with the other environmental factors we measured, regardless of the time lag (Table 2). The weak impact of air temperature, VPD, and soil moisture on δ13Ceco in our study reflects the weak link between assimilation and respiration at a seasonal scale. Additionally, stronger correlations were reported by Schaeffer et al. [65] after they separately calculated the δ13Ceco using data within or below the canopy by selectively removing periods when the air within and below the canopy was well mixed. These findings suggest that the environmental controls for δ13Ceco may also be influenced by other factors, such as the canopy's aerodynamic properties.

Although we found different seasonal isotopic patterns in different respiratory C pools and different environmental factors can contribute differently to the δ13CR of those pools over the entire growing season, some strong correlations were still observed between the δ13CR of the leaf, trunk, and soil and the δ13Ceco. The most obvious occurred in May, when the δ13C from respiratory C pools as well as the ecosystem showed decreasing trends (Figure 4). These distinct decreases may have resulted from unique extreme weather events in May, when the ecosystem received a large amount of global radiation but less rainfall, which led to an overall increase in VPD, a decrease in soil moisture, and a large temperature difference between the air and the soil (Figure 2). The concurrent decrease in δ13CR and δ13Ceco suggests that although the linkage between assimilates and respiration was weak at a seasonal scale in our study, recently fixed assimilates could be allocated to the location of respiration

in a short period of time, indicating a quick link between different respiration pools and assimilation at the beginning of the growing season in the coniferous and broad-leaved mixed forest.

#### **5. Conclusions**

Overall, the observed difference in seasonal variation in photosynthetic discrimination between the deciduous broadleaved and evergreen needle species in our forest indicated a remarkable difference in C assimilation between them. As expected, post-photosynthetic discrimination explained the substantial isotopic differences among C pools and their respiratory fluxes during the process of photosynthate allocation and respiration. Compared to leaf and soil δ13CR, trunk δ13CR was more sensitive to environmental changes and related to the seasonal patterns of leaf δ13C, which suggests that trunk δ13CR has great potential as a way to record discrimination-encoded biochemical and physiological information related to C cycling. In addition, the linkages between environmental factors and δ13Ceco were not constant, which complicates the use of δ13Ceco to determine the linkage between assimilation and respiration. We conclude that the effects of environmental changes on C isotope discrimination can be distracted through a number of processes during C transfer.

We suggest that the combined effect of multiple environmental factors and the potential time lags should be addressed carefully when evaluating the long-term temporal changes of δ13CR and δ13Ceco. Apart from the biological impacts initiated by controlling the photosynthetic rate and stomatal openness, the physical parameters, such as aerodynamic conditions (e.g., atmospheric stability) and the conditions of the soil–atmosphere diffusion systems may also regulate δ13Ceco, although they seem to act as distracters, impairing the relationship between environmental factors and δ13Ceco. Furthermore, soil δ13CR and δ13Ceco integrate the δ13C signals of multiple tree species with different C assimilation and transfer patterns in mixed forests. In these respects, the present study expands our knowledge of C isotope discrimination in mixed forests with multiple tree species and effectively promotes a comprehensive consideration of the influencing factors related to isotopic variation within process-based ecosystem models.

**Author Contributions:** J.W. designed the study; H.D. performed the research; A.W., F.Y. and G.D. provided the resources; H.D., D.G. and J.W. contributed to the data analysis; D.G. and J.W. contributed to the funding acquisition; H.D. wrote the first draft and all authors contributed to the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (grant numbers 31870625, 41975150).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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