**1. Introduction**

Stable carbon (C) isotope measurement is a useful tool for disentangling C cycling processes in forest ecosystems, as a unique C isotopic signature is imprinted along the C transfer pathway from CO2 in the atmosphere to C in different ecosystem pools, which is affected by isotopic discrimination (Δ). During photosynthesis, C3 plants discriminate heavily against 13C through photosynthetic discrimination; the resulting C isotopic signature (δ13C) in the photosynthate is more negative compared to the δ13C of atmospheric CO2 (δ13Cair) and is linearly dependent on the ratio of the intercellular and atmospheric CO2 concentration (ci/ca) [1,2]. After photosynthesis, plants

rapidly release photo-assimilated C into the soil, and the post-photosynthetic discrimination effects become involved in a number of biochemical processes alongside mixing and exchange with other C storages, which leads to different δ13C levels among woody plant parts and soil [3,4]. Subsequently, respiration-associated discrimination and the consumption of substrates with different δ13C values further modify the δ13C of respiratory fluxes, resulting in apparent differences between the δ13C of respired CO2 (δ13CR) and the respiration substrates [5–8]. The accurate measurement of δ13C in different transfer pathways is important to explore the complexity of the C cycle under the changing climate.

Moreover, the discrimination processes encode biochemical and physiological information into δ13C, and this information is transferred through different stages of the C cycle. Thus, thoroughly investigating δ13C in each C pool and respiratory flux can help evaluate the coupling between each of the processes in the pathways of C cycling. For example, strong evidence of the linkage between plant photosynthesis and soil respiration was found by Kuzyakov and Gavrichkova [9], indicating that the δ13C of soil-respired CO2 may also be related to photosynthetic discrimination via root respiration or exudation. Ecosystem respiration combines the CO2 emitted from leaves, trunks, and soil within the ecosystem, and it also integrates their C isotopic signals. Thus, the δ13C in ecosystem respiration (δ13Ceco) can vary substantially with changes in the relative contribution of the respiration of each component.

Discrimination also encodes information about the dynamics of environmental drivers into δ13C. Seasonal variation in environmental factors, such as vapor pressure deficit (VPD) and soil moisture, influence stomatal conductance, which then alters photosynthetic discrimination and further leads to seasonal dynamics in the δ13C of CO2 respired from leaves [7]. Due to the linkages between the photosynthetic metabolites and the substrates of different respiratory fluxes, several studies also found that changes in environmental drivers may be responsible for seasonal dynamics in the δ13CR of trunks, roots, and soil [10–12]. Accordingly, the δ13C of ecosystem respiration, which integrates all respiratory components of the ecosystem, was also found to have significant short-term and seasonal variations [13–15], which have been directly or implicitly linked to environmental factors with a time lag. This indicates either a fast linkage between assimilates and ecosystem respiration or a linkage delayed by the time in which the assimilates are transported to the sites of respiration [10,16].

However, a significant response of δ13Ceco to environmental factors does not always exist. For example, McDowell et al. [13] found only a weak relationship between canopy conductance and δ13Ceco over a 2-week period, which they attributed to noncanopy controls, such as belowground respiration on δ13Ceco. Similarly, the results of long-term measurements with high time resolution showed that δ13Ceco was not tightly coupled with environmental factors in some of the study periods [17]. These studies collectively indicate that the response of δ13Ceco to environmental changes is highly variable and that the influence of complicated environmental conditions, combined with other factors, may weaken the linkage between assimilates and ecosystem respiration. Thus, there is a practical need to explore what factors control δ13Ceco and to what extent δ13Ceco reveals the links between C cycling processes.

Photosynthetic discrimination can be calculated at the leaf level by determining the difference between the δ13C of leaf organic matter and δ13Cair [1], or it can be measured online using branch chambers [17]. Alternatively, photosynthetic discrimination can be estimated using the comprehensive Farquhar's model at a canopy scale by combining δ13C and CO2 flux measurements [18,19]. On the other hand, respiration-associated discrimination can be evaluated by chamber measurements [20,21] using the Keeling plot approach [22,23]. This approach is based on the theory of the two-component gas-mixing model, where δ13CR emerges as the y-intercept of the mixing relationship between δ13C and the inverse of the CO2 concentration (CO2) [24,25]. The Keeling plot approach can also determine the δ13Ceco using nighttime CO2 and δ13C across the vertical profile of the forest [14,17]. Recently, optical laser spectroscopy techniques have allowed in situ continuous monitoring of CO2 isotopes within open forest canopies or closed gas exchange chambers, thus tracing the high-resolved dynamics in δ13Cair, δ13CR, and δ13Ceco over more weather conditions [12,17]. However, few studies have

simultaneously measured δ13Cair, δ13CR, and δ13Ceco, which represents a limitation of our knowledge on discrimination in the process of C exchange.

Coniferous and broad-leaved mixed forests are widely distributed in northeast China. These forests are well-protected old-growth virgin forests characterized by rich species diversity as well as high biomass. Previous studies have shown that coniferous and broad-leaved mixed forests are strong C sinks and have experienced climate warming over the past 50 years [26]. To our best knowledge, variations of δ13C in forest storage C pools and respiratory fluxes have mainly been studied in pure forests [17,27–29], while mixed forests with multiple tree species have rarely been covered by previous studies [12]. In the present study, we continuously investigated in situ seasonal variations in the δ13Cair and δ13CR of leaves, trunks, and soil as well as δ13Ceco in a 200-year-old coniferous and broad-leaved mixed forest over a full growing season. We also explored the Δ variation of the dominant species in this mixed forest. Firstly, we aimed to identify species-specific differences in photosynthetic Δ and variations in the Δ subsequent photosynthesis in this mixed forest. Secondly, we assessed the environmental effects on seasonal variations in δ13C for CO2 respired by leaves, trunks, and soil as well as the ecosystem. Finally, we tested whether δ13Ceco reveals links between aboveground and belowground processes.

#### **2. Materials and Methods**

#### *2.1. Site Description*

The study was carried out in a coniferous and broad-leaved mixed forest site (42◦ 24.149 N, 128◦ 05.768 E, 732 m elevation) in the Changbai mountain natural reserve, Jilin, northeast China. The site is pristine and undisturbed by anthropic activities and characterized by richness of species in both its overstorey and understorey. The density of the trees is about 560 trees ha−<sup>1</sup> (stem diameter > 8 cm). Dominant species include one evergreen needle species, Korean pine (*Pinus koraiensis*), and four deciduous broadleaf species, including Manchurian ash (*Fraxinus mandshurica*), Mono maple (*Acer mono*), Mongolian oak (*Quercus mongolica*), and Tuan linden (*Tilia amurensis*). *P. koraiensis*, *F. mandshurica*, and *T. amurensis* account for about 40%, 30%, and 20% of the total tree number, respectively, while *Q. mongolica* and *A. mono* account for the remaining 10%. The average age of the dominant species has been estimated to be over 200 years, and the mean canopy height is about 26 m. The growing season of the vegetation extends from May to September. The site's topography is nearly flat. The soil is classified as dark brown forest soil with a depth of 60–100 cm. The climate is temperate, with a mean annual temperature of 3.6 ◦C and annual precipitation of 695.3 mm measured between 1982 and 2003; over 80% of the rainfall occurs within the growing season.
