Forest Ecosystems 10 (2023) 100123 Contents lists available at ScienceDirect Forest Ecosystems journal homepage: www.keaipublishing.com/cn/journals/forest-ecosystems Ecological stoichiometric comparison of plant-litter-soil system in mixed-species and monoculture plantations of Robinia pseudoacacia, Amygdalus davidiana, and Armeniaca sibirica in the Loess Hilly Region of China ~uelas d, e Senbao Lu a, b, Yunming Chen a, c, *, Jordi Sardans d, e, Josep Pen a State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling, 712100, China b University of Chinese Academy of Sciences, Beijing, 100049, China c Institute of Soil and Water Conservation, Northwest A&F University, Yangling, 712100, China d CSIC, Global Ecology Unit CREAF-CSIC-UAB, Bellaterra, 08193, Catalonia, Spain e CREAF, Cerdanyola del Valles, 08193, Catalonia, Spain A R T I C L E I N F O A B S T R A C T Keywords: We examined how afforestation patterns impact carbon (C), nitrogen (N), and phosphorus (P) stoichiometry in the Stoichiometry plant-litter-soil system. Plant leaf, branch, stem, and root, litter, and soil samples were collected from mixed- Plant species plantations of Robinia pseudoacacia with Amygdalus davidiana (RPAD), R. pseudoacacia with Armeniaca Litter sibirica (RPAS), and monocultures of R. pseudoacacia (RP), A. davidiana (AD), and A. sibirica (AS) in the Loess Hilly Soil Region. The results showed that in mixed-species plantations, R. pseudoacacia had lower leaf N and P concen- Tree mixture Loess Plateau trations than in monocultures, while both A. davidiana and A. sibirica had higher leaf N and P concentrations. Soil P limited tree growth in both afforestation models. Mixing R. pseudoacacia with A. davidiana or A. sibirica reduced N-limitation during litter decomposition. Average soil total N and P concentrations were higher in RPAS than in RPAD, and both were higher than the corresponding monocultures. The average soil C:N ratio was the smallest in RPAS, while the average soil C:P ratio was larger in RPAS than in RP. A positive correlation between N and P concentrations, and between C:N and C:P ratios, was found in litter and all plant organs of mono- and mixed- stands. Alternatively, for N concentration and C:N ratio, the correlations between plant (i.e., leaf, branch, root) and litter and between plant and soil were inverse between plantation types. RPAD has an increased litter decomposition rate to release N and P, while RPAS has a faster rate of soil N mineralization. RPAD was the best plantation (mixed) to improve biogeochemical cycling, as soil nutrient restrictions, particularly for P-limitation, on trees growth were alleviated. This study thus provides insights into suitable tree selection and management by revealing C:N:P stoichiometry in the plant-litter-soil system under different afforestation patterns. 1. Introduction complementarity effect with respect to corresponding monocultures (Khanna et al., 2008). To preserve existing plantations and create new Plantations in the Loess Hilly Region are divided into mixed-species plantations, it is often desirable to convert monocultures into species-rich and monoculture plantations. Mixed-species plantations consist of two mixed plantations, as it is frequently observed that the stability and or more species of trees. Generally, mixing suitable tree species can sustainability of mixed-species plantations is significantly higher than improve soil fertility, regulate microclimate, and reduce the occurrence that of monocultures (Forrester et al., 2006). However, the sustainable and spread of pests, diseases, and fires (Camp, 1986; Coll et al., 2018). development of mixed-species plantations depends not only on the in- Previous studies have shown that mixed-species plantations promote dividual characteristics of tree species (Wojciech et al., 2019), but also on nutrient circulation via canopy differentiation, root stratification, and the site conditions, such as soil nutrient status (He and Dijkstra, 2014; * Corresponding author. State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling, 712100, China. E-mail address: ymchen@ms.iswc.ac.cn (Y. Chen). https://doi.org/10.1016/j.fecs.2023.100123 Received 12 January 2023; Received in revised form 5 June 2023; Accepted 5 June 2023 2197-5620/© 2023 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S. Lu et al. Forest Ecosystems 10 (2023) 100123 Sardans et al., 2017). Thus, it is difficult to predict the species compo- that plants submitted to drought in a mesic European shrublands sitions that would improve the stability of mixed-species plantation increased their foliar C:N and C:P ratios associated with a metabolic shift (Forrester et al., 2014). towards over production of C-rich secondary metabolites linked to Carbon (C), nitrogen (N), and phosphorus (P) stoichiometry focuses anti-stress mechanisms, whereas decreased their root C:N, C:P, and N:P on the interactions and balance of chemical elements in the biogeo- ratios linked to an increase of metabolism. Therefore, the analysis of chemical process (Sterner and Elser, 2002; Güsewell, 2004). It is also stoichiometric changes in the distinct compartments of the plant-soil used to provide a scientific approach to study plant growth and nutrient system is warranted to obtain an overall view of the plant-soil system limitations in a terrestrial ecosystem (Pe~ nuelas and Baldocchi, 2019). response to environmental changes from a stoichiometric point of view Previous studies have analyzed the C:N:P stoichiometry of plants from a and relate them to ecophysiological responses. regional to global scale, in mixed-species plantations, to uncover nutrient To address this knowledge gap, we investigated C:N:P stoichiometric cycling, limitations, and feedback of multiple tree species (Yang et al., of plant organ (i.e., leaf, branch, stem, root), litter, and soil samples in 2018; Hou et al., 2018). Few studies have revealed the plant C:N:P mixed-species and monoculture plantations of R. pseudoacacia, A. stoichiometry discrepancy between mixed-species plantations and their davidiana, and A. sibirica in the Loess Hilly Region. Our concrete aims corresponding monocultures. Existing studies have focused mainly on a were to understand: (1) whether mixed-species plantations changed the few organs, such as the leaf (Jiao et al., 2022) and fine root (Wu et al., C:N:P stoichiometric of plant organs, litter, and soil compared to mono- 2021b). Additionally, differences in C:N:P stoichiometry among tree cultures; (2) whether litter C:N:P correlated with the C:N:P stoichiometry species in different plantations can reflect the relationship of plant of plant organs or soil; and (3) whether soil C:N:P correlated with the nutrient status with biogeochemical processes in the community (Dawud C:N:P ratio of other plant organs. We hypothesized that (1) the dynamic et al., 2016). Thus, C:N:P stoichiometry provides an opportunity to un- concentrations of C:N:P stoichiometry in the plant-litter-soil system may derstand plant nutrient relationships in mixed-species plantations. be influenced by different tree species, afforestation patterns, or the plant Soil is the primary source of nutrients for plant growth. Soil nutrient organ and soil layer used for analysis; (2) the correlations between C, N, concentrations are affected by litter, organic matter, and soil microor- and P concentrations and ratios would be all positive or negative in plant ganisms (Richard et al., 2013). Studies on mixed plantations of Hippophae organs, litter, and soil of different plantations; and (3) the leaf, branch, rhamnoides reported a strong relationship between leaf and litter C:N:P and fine root composition of N and P would be derived from litter stoichiometry, and the correlation between N and P concentrations was decomposition, while the C:N:P stoichiometry of stem may be more more significant among leaf, litter, and soil than those corresponding to C related to bulk soil. concentration (Wu et al., 2021b). Jiao et al. (2022) found that P con- centration and C:P ratio were significantly correlated in litter and soil in 2. Materials and methods mixed-species plantations. Chapin et al. (2011) found that 90% of the N and P elements released from litter into the soil were reabsorbed and 2.1. Site description utilized by plants. Pang et al. (2021) studied the C:N:P stoichiometry of secondary mixed forests in the Qinling Mountains and found a close This study was performed in Ansai County (36 350 4900 –36 350 5600 N, relationship in the plant-litter-soil continuum. Thus, litter influences the 109 130 1800 –109 130 3200 E, with an average elevation of 1,450 m), chemical and physical properties of soil, and microbial activities through located on the interior of the Loess Plateau (Fig. 1a). This region has a the release of organic carbon and nutrients, and can indirectly affect warm, temperate, semi-arid, continental monsoon climate, with an nutrient allocation to distinct organs via plant-soil nutrient cycling annual mean temperature of 8.8  C. The maximum and minimum tem- (Chapman and Newman, 2010; Yang et al., 2018). peratures are 36.8 and 23.6  C, respectively. The mean annual pre- The Loess Plateau is one of the most eroded loess regions in the world. cipitation is 500 mm, with the rainy season (June–September) To reduce water loss and soil erosion, the Chinese government imple- accounting for 72.9% of the total annual rainfall. Soils in this area are mented the "Grain to Green" project in the 1950's (Feng et al., 2016). To classified as Calcic Cambisols (FAO, 2020). Excessive logging and date, the area covered by plantation (7.5  104 ha) accounts for 60% of reclamation in the study area have resulted in less natural forest, and an the total area (Liu et al., 2017). Due to its high growth rate and ability to increase in artificially created landscapes (Fu et al., 2017). Among local improve soil nutrient conditions via N2-fixing (Shan et al., 2002), Robinia forest plantations, the primary tree species are R. pseudoacacia, Pinus pseudoacacia (>7.0  104 ha) has become the most important introduced tabuliformis, Platycladus orientalis, Ulmus pumila, A. davidiana, and A. tree species for afforestation in this region (Cao and Chen, 2017). sibirica. Shrub species include H. rhamnoides and Caragana intermedia. However, at half-maturity (19–25 years), the R. pseudoacacia community Herb species include Medicago sativa, Herba artemisiae, Stipa bungeana, displayed growth slowdown and canopy wilting, implying reduced Bothriochloa ischaemum, and Artemisia gmelinii. ecosystem services (Wei et al., 2018). Amygdalus davidiana and Arme- niaca sibirica, two native tree species in the Loess Hilly Region, possess 2.2. Experimental design drought-resistant, well-developed roots and strong barberries. In addi- tion, the kernel of A. davidiana is often made for decoration, while the Sampling sites were selected in mixed-species plantations that pulp of A. sibirica is edible. To conserve soil and water and improve included mixed-species plantations of R. pseudoacacia with A. davidiana economic benefits, these two trees are widely cultivated with (RPAD), R. pseudoacacia with A. sibirica (RPAS) and in monocultures of R. pseudoacacia. Previous studies primarily focused on the plant and soil R. pseudoacacia (RP), A. davidiana (AD), and A. sibirica (AS) at half- C:N:P stoichiometry of R. pseudoacacia plantations (Cao and Chen, 2017; maturity (19–25 years) with normal growth and similar structures Zhang et al., 2019); however, the correlation of C:N:P stoichiometry (Fig. 1b). In April 2021, three 20 m  20 m plots (separated by more than among the plant-litter-soil system of monocultures and mixed-species 100 m) were established in each of five different plantations. Coordinate plantations have been rarely evaluated. In this context, several studies and elevation information for the sampling sites was marked as experi- have shown that soil and litter nutrient characteristics together influence mental plot sites using Ovey map (V.9.3.0., Beijing, China) (Table 1). the nutrient characteristics of plant (Chapin et al., 2011). The intrinsic Topographic factors (i.e., gradient, aspect) were determined with a hand- connections between plant nutrients stoichiometry and the nutrient held GPS compass (JSD-X2., Beijing, China) (Table 1). Stand structures of characteristics of litter can improve our understanding of plant nutrient all tree individuals in each plot were investigated (Table 2), including limitations and ecosystem dynamics, according to stoichiometry theory. stand density (SD), crown area (CA), average tree height (AH), diameter An overall view of plant stoichiometry shifts to environmental changes is at breast height (DBH), and height under branch (HB) (Table 2). necessary to discern the stoichiometric changes of the most plant organs as possible. For example, Gargallo-Garriga et al. (2014, 2015) observed 2 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 1. Sample plot (b) is located on the Loess Plateau of China (a). RPAD: R. pseudoacacia and A. davidiana mixed plantation; RPAS: R. pseudoacacia and A. sibirica mixed plantation; RP: R. pseudoacacia monoculture; AD: A. davidiana monoculture; and AS: A. sibirica monoculture. Table 1 Basic information for the sample sites and soil characteristics of mixed-species and monoculture plantations. Afforestation pattern Plantation type Dominant species No. Longitude (N) Latitude (E) Altitude (m) Gradient ( ) Aspect Mixture RPAD R. pseudoacacia þ A. davidiana 1 109 130 42.5600 36 350 52.0300 1189 20.4 Semi-sunny 2 109 130 27.2700 36 350 29.3500 1191 29.2 Semi-sunny 3 109 130 12.2200 36 350 30.8200 1199 22.6 Semi-sunny RPAS R. pseudoacacia þ A. sibirica 1 109 130 50.8800 36 350 21.1000 1195 22.3 Semi-sunny 2 109 130 45.1800 36 350 34.1700 1142 23.9 Semi-sunny 3 109 130 36.8700 36 350 50.7400 1203 20.4 Semi-sunny Monoculture RP R. pseudoacacia 1 109 130 39.7200 36 350 50.8500 1190 21.4 Semi-sunny 2 109 130 56.4600 36 350 24.8800 1212 21.7 Semi-sunny 3 109 130 19.8400 36 350 29.7000 1180 14.0 Semi-sunny AD A. davidiana 1 109 130 36.8300 36 350 50.3200 1173 28.8 Semi-sunny 2 109 130 39.3600 36 350 47.7200 1157 25.0 Semi-sunny 3 109 130 43.4300 36 350 49.1900 1160 21.0 Semi-sunny AS A. sibirica 1 109 130 50.2800 36 350 57.5900 1160 24.0 Semi-sunny 2 109 130 56.9000 36 350 48.9600 1222 18.6 Semi-sunny 3 109 160 31.8500 36 360 40.0200 1224 17.3 Semi-sunny Notes: RPAD, R. pseudoacacia and A. davidiana; RPAS, R. pseudoacacia and A. sibirica; RP, R. pseudoacacia; AD: A. davidiana; AS, A. sibirica. Table 2 Descriptive statistics of tree growth and soil properties among mixed-species and monoculture plantations. Afforestation Plantation Dominant SD (trees⋅ha1) CA (m2) AH (m) DBH HB (m) BD SWC (%) EC pH pattern type species (cm) (g⋅cm3) (mS⋅cm1) Species Total Mixture RPAD R. pseudoacacia 1542  1891  262.14  8.37  9.35  3.41  1.25  11.13  107.57  8.58 213 a 150 A 46.86 0.23 ab 0.26 0.19 ab 0.05 A 0.63 A 0.98 A  0.12 A. davidiana 350  92.67  4.72  6.01  1.08  63 b 15.48 b 0.29 a 0.30 0.08 a RPAS R. pseudoacacia 792  1167  215.86  8.09  9.02  3.03  1.25  9.91  107.33  8.52 122 b 101 B 54.42 0.50 b 0.81 0.24 b 0.04 A 0.08 B 1.11 A  0.06 A. sibirica 375  93.94  5.96  9.62  0.57  109 b 14.21 0.25 a 0.41 0.07 a Monoculture RP R. pseudoacacia 1075  1075  190.04  9.63  11.67 3.84  1.24  9.44  105.50  8.66 76 ab 76 B 2.87 0.39 a  1.11 0.23 a 0.05 A 0.06 B 0.60 A  0.01 AD A. davidiana 733  733  143.17  3.58  5.65  0.37  1.19  10.54  82.30  8.72 33 a 33 C 5.47 a 0.12 b 0.64 0.05 b 0.05 B 0.08 AB 0.76 C  0.03 AS A. sibirica 700  700  126.66  3.46  10.09 0.34  1.27  7.91  87.95  8.72 58a 58 C 7.23 0.05 b  0.77 0.04 b 0.05 A 0.19 C 0.81 B  0.03 Notes: RPAD, R. pseudoacacia and A. davidiana; RPAS, R. pseudoacacia and A. sibirica; RP, R. pseudoacacia; AD: A. davidiana; AS, A. sibirica. SD, stand density; CA, crown area; AH, average tree height; DBH, diameter at breast height; HB, height under branch; BD, bulk density; SWC, soil water content; EC, electric conductivity. Different uppercase letters above the bars indicate significant differences among different plantation types, while different lowercase letters indicate significant differences among different plantation types for the same tree species (P < 0.05). 3 S. Lu et al. Forest Ecosystems 10 (2023) 100123 2.3. Sample collection 2.5. Statistical analysis In August 2021, five target trees with similar height and DBH were All data was subject to the one-sample Kolmogorov-Smirnov test, selected for sampling in each plot. Three branches with the same diam- which subsequently met the requirements for normal distribution. A one- eter (5 mm) were collected from the upper, middle, and lower canopies in way ANOVA and Duncan test were used to compare the discrepancy of order to collect intact mature leaf samples without pest or disease dam- C:N:P stoichiometry in plant, litter, and soil among mixed-species and age. To reduce the effects of asymmetric tree trunk growth, stem samples monoculture plantations. A t-test was used to compare the discrepancies were taken from both directions (east-west and north-south) of the trunk between C:N:P stoichiometry for the same species in different planta- using growth cones (diameter ¼ 0.5 cm). Since no suitable allometric tions. A principal component analysis (PCA) and Pearson correlation equation can simultaneously calculate the biomass of all three trees, a coefficient were used to determine the correlations within the C:N:P universal coefficient of 0.5 was used to represent carbon storage in stoichiometry in the plant-litter-soil system. A linear mixed model various organs of different tree species in mixed-species plantations ANOVA was used to determine the effects of tree species (TS) and (Zeng et al., 2018). A biomass weighted average method was used to afforestation pattern (mixed versus mono, AP) on the C:N:P stoichiom- calculate the C, N, and P concentrations of each organ in mixed-species etry of litter, as well as to determine the effects of the average soil, TS, AP, plantations, with the calculation formula as follows (Liu et al., 2019): and plant organ (PO) on the plant C:N:P stoichiometry. The effects of TS, P AP, and soil layer (SL) on the soil C:N:P stoichiometry was also assessed. Ei  Bi Linear regression analysis was used to determine the relationships of Eorg ¼ P (1) Borg C:N:P stoichiometry among plant, litter, and soil, as well as between plants and their contributing factors, i.e., litter or soil. Variation parti- where Eorg is organ C, N, and P concentrations of mixed-species planta- tioning analysis (VPA) was used to determine the contribution of litter tions; Ei and Bi are the organ C, N, and P concentrations and biomass of and soil to the plant C:N:P stoichiometry in different plantations. SPSS each tree species, respectively; and Borg is the organ biomass of all tree 18.0 (IBM Inc., New York, USA) was used to analyze the data. VPA and species in the mixed-species plantations. PCA were performed using Canoco 5 (Ter Braak and Smilauer, 2012). Nine sample points were established along an S-shape in each plot, Figures were created using Origin 2017 software (Originlab Inc., USA). and a root drill (diameter ¼ 9 cm) was used to obtain fine root (diameter The significance level was set at P < 0.05. <2 mm) samples at 0–20, 20–40, and 40–60 cm. Soil samples were collected simultaneously. In addition, single soil samples at 0–20 cm 3. Results depth profile were collected from each plot using 100 cm3 ring knife. To avoid the effect of fine root and litter on the sample, the soil organic layer 3.1. Plant growth and soil properties and debris was removed prior to sampling. In mid-November 2021, freshly fallen and not yet decomposed leaf litter was collected. Nutrient As shown in Table 2, the afforestation pattern had no significant effect on composition from a soil depth of 0–60 cm was calculated using the CA, DBH, or pH (P > 0.05). Compared to monocultures, SD was smaller, following equation: while AH, and HB were larger for A. davidiana and A. sibirica in mixed P plantations (P < 0.05); SD and AH did not change significantly for E  BDk  Tk Esoil ¼ Pk (2) R. pseudoacacia in mixed plantations (P > 0.05), while HB was larger for BDk  Tk R. pseudoacacia in RPAD (P < 0.05). Similarly, total SD was larger (P < 0.05), where Esoil is C, N, and P concentrations in 0–60 cm soil layer, Ek is C, N, while AH and HB did not change significantly for R. pseudoacacia in RPAD and P concentrations in k soil layer, BDk is bulk density in k soil layer, and than in RPAS (P > 0.05). In addition, SWC was larger (P < 0.05), while BD Tk is depth in k soil layer. and EC did not change significantly in RPAD than in RPAS (P > 0.05). 2.4. Laboratory analyses 3.2. Plant C:N:P stoichiometry Plant and litter samples were oven-dried at 60  C for 48 h to constant Fine root N and P concentrations of R. pseudoacacia, leaf and branch N weight (Yang et al., 2018), and subsequently ground up and sieved and P concentrations of A. davidiana were higher in RPAD than in RP and (diameter ¼ 0.25 mm). Soil samples were air-dried and passed through AD, leaf N concentration, root P concentration of R. pseudoacacia, leaf 2-mm mesh sieves to remove large roots and stones. They were then and branch N concentrations, stem N concentration of A. sibirica were ground and sieved through 0.15-mm mesh to measure soil organic car- higher in RPAS than in RP and AS (Table 3). In addition, R. pseudoacacia bon (SOC), total nitrogen (TN), and total phosphorus (TP) (Pang et al., in RPAD had higher branch and stem C, N and P concentrations than 2021). The C concentration of plant, litter, and soil (organic carbon) R. pseudoacacia in RPAS, while leaf and root C, N and P concentrations samples was determined via oil bath K2Cr2O7 titration (Nelson and were in the other way around. Sommers, 1982). Organ, litter, and soil samples were digested with At the same time, C:P ratio of stem and branch, N:P ratio of fine root H2SO4–H2O2 and H2SO4–HCIO4. N and P concentrations were then of R. pseudoacacia, N:P ratio of leaf and branch, C:P and N:P ratios of stem determined using the semi-automatic Kjeldahl method (Bremner and and root of A. davidiana were higher in RPAD than in RP and AD, branch, Mulvaney, 1982) and colorimetric method (Murphy and Riley, 1962), and stem C:N ratios of R. pseudoacacia, leaf and branch N:P ratios, stem respectively. Soil bulk density (BD) and soil water content (SWC) were N:P ratio of A. sibirica were higher in RPAS than in RP and AS (Table 4). measured using the cutting ring method (Bao, 2000), soil pH was In addition, R. pseudoacacia in RPAD had lower C:N and C:P ratios of leaf, determined using an automatic acid–base titrator (PB-10 standard pH branch, stem and root than R. pseudoacacia in RPAS, while the N:P ratio meter; Sartorius, G€ ottingen, Germany) with a water:soil ratio of 2.5:1, of leaf, branch, stem, and root was in other way around. and electric conductivity (EC) was determined using a conductivity meter Several factors were found to have significant effects on plant C:N:P (DDSJ-308F, INESA Scientific InstrumentCo., Ltd, Shanghai, China). stoichiometry, including tree species, plant organ interactions, and Notes: roots (and all the organs) of each species were analyzed as sepa- interaction of tree species, plant organ, and afforestation pattern (P < rate samples and not mixed samples of the two species previous to ana- 0.05, Table S1). Tree species, afforestation pattern, and plant organ in- lyses to determine C, N and P. teractions had significant effects on plant C concentration and C:P ratio (P < 0.01, Table S1). 4 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Table 3 Characteristics of the C, N and P concentrations in plant (leaf, branch, stem, and fine root) among mixed-species and monoculture plantations. Data are means  SE. Element and their Plant Pure plantation Mixed plantation ratios organ R. pseudoacacia A. davidiana A. Sibirica R. pseudoacacia þ A. davidiana R. pseudoacacia þ A. Sibirica C Leaf 470.00  0.46 Db 481.60  0.94 477.16  0.75 455.54  1.94 456.90  0.90 489.42  1.09 446.41  0.33 Ba Ca Dc Eb Ba Eb Branch 482.58  1.23 Ca 463.64  1.67 462.47  0.54 483.88  2.63 496.42  1.21 470.07  0.90 457.03  1.07 Db Ea Ca Ba Db Db Stem 492.64  1.81 Ba 471.88  1.06 503.30  0.47 492.95  2.64 481.11  0.52 477.22  0.61 482.83  0.72 Cb Ba Ba Da Cb Cb Root 510.12  3.47 Aa 502.23  1.41 546.47  0.84 505.09  1.56 507.90  1.11 508.54  1.39 534.94  0.96 Aa Aa Aa Aa Aa Ab N Leaf 28.21  0.12 Aa 17.40  0.37 Ab 13.10  0.07 Ab 27.76  0.23 Ba 25.45  0.15 Aa 26.56  0.20 Ab 16.73  0.26 Aa Branch 10.11  0.15 Da 6.56  0.08 Db 4.50  0.06 Bb 10.40  0.29 Da 7.38  0.19 Da 8.52  0.27 Db 5.22  0.13 Da Stem 3.35  0.12 Ea 3.52  0.09 Ea 2.51  0.05 Ea 3.56  0.21 Ea 2.29  0.07 Eb 2.18  0.10 Eb 1.88  0.03 Eb Root 18.54  0.13 Bc 7.52  0.08 Cb 4.11  0.09 Cb 29.93  0.27 Aa 9.55  0.24 Ca 20.14  0.47 Bb 6.37  0.07 Ca P Leaf 1.21  0.04 Aa 0.95  0.06 Ab 0.84  0.01 Ab 0.95  0.05 Ab 1.15  0.02 Aa 1.19  0.04 Aa 1.01  0.03 Aa Branch 0.43  0.01 Bc 0.46  0.01 Cb 0.37  0.00 Ba 0.52  0.00 Ba 0.50  0.01 Ca 0.47  0.01 Bb 0.34  0.03 Ca Stem 0.13  0.02 Da 0.21  0.01 Da 0.11  0.01 Ea 0.11  0.01 Cab 0.09  0.00 Eb 0.09  0.01 Db 0.10  0.01 Db Root 0.39  0.01 BCc 0.39  0.02 Ca 0.21  0.01 Db 0.56  0.01 Ba 0.40  0.02 Da 0.47  0.01 Bb 0.39  0.01 BCa Notes: Different uppercase letters above the bars indicate significant differences among different plant organ for the same plantation type, while different lowercase letters indicate significant differences among different plantation type for the same plant organ (P < 0.05). Table 4 Characteristics of the C:N, C:P, and N:P ratios in plant (leaf, branch, stem, and fine root) among mixed-species and monoculture plantations. Data are means  SE. Element and their Plant Pure plantation Mixed plantation ratios organ R. pseudoacacia A. davidiana A. Sibirica R. pseudoacacia þ A.davidiana R. pseudoacacia þ A. Sibirica C:N Leaf 16.66  0.06 Db 27.71  0.55 Da 36.42  0.14 Ea 16.41  0.08 Cb 18.11  0.07 Eb 18.43  0.10 Ca 26.70  0.40 Db Branch 47.74  0.60 Bb 70.68  0.62 Ba 102.90  1.32 46.57  1.03 Bb 67.33  1.61 Ba 55.28  1.63 Ba 87.61  1.93 Bb Da Stem 147.59  4.86 Ab 134.05  2.96 200.37  4.04 139.38  8.06 Ab 210.31  6.51 219.73  9.56 256.62  3.77 Ab Ab Aa Aa Aa Root 27.51  0.00 Ca 66.82  0.53 Ba 133.10  2.61 16.88  0.10 Cc 53.25  1.23 Cb 25.27  0.54 Cb 83.99  0.83 Bb Ca C:P Leaf 390.55  13.06 Cb 508.36  29.70 567.26  9.12 Ea 480.04  24.11 398.01  5.94 410.86  11.08 443.40  12.28 Da Ca Db Cb Cb Branch 1130.48  35.54 1010.17  9.65 1251.10  936.14  3.35 Bc 997.25  15.36 1008.09  1360.52  Ba Ca 12.44 Da Bb 15.94 Bb 126.58 Ba Stem 3915.18  487.00 2304.35  68.16 4672.95  4515.97  5623.78  5648.60  4967.57  Ab Ab 242.43 Aa 207.46 Aab 177.36 Aa 407.44 Aa 297.64 Aa Root 1323.97  32.51 1280.19  68.21 2656.50  92.46 896.64  16.22 1262.79  1095.35  1385.64  Ba Ba Ba Bc 67.92 Ba 29.62 Bb 36.80 Bb N:P Leaf 23.44  0.72 Bb 18.32  0.74 15.57  0.19 Cb 29.25  1.38 Ca 21.98  0.25 Ba 22.29  0.49 Db 16.60  0.28 Ba ABb Branch 23.67  0.59 Ba 14.29  0.15 Ca 12.16  0.04 Db 20.12  0.46 Db 14.82  0.21 Da 18.25  0.31 Ec 15.48  1.11 Ba Stem 26.38  2.40 Bab 17.19  0.41 Bb 23.30  0.81 Aa 32.51  1.61 Ca 26.74  0.24 Aa 25.66  1.06 Cb 19.34  0.93 Ab Root 48.12  1.17Ab 19.15  0.88 Ab 19.95  0.32 Ba 53.12  0.68 Aa 23.69  0.90 Ba 43.33  0.37 Ac 16.49  0.28 Bb Notes: Different uppercase letters above the bars indicate significant differences among different plant organ for the same plantation type, while different lowercase letters indicate significant differences among different plantation type for the same plant organ (P < 0.05). 3.3. Litter and soil C:N:P stoichiometry (Fig. 3d–f). In addition, shallow SOC concentration, soil C:N, and C:P ratios were higher in RPAD than in RPAS (Fig. 3a, d–e), while shallow Litter C concentration and C:P ratio was smaller in RPAD than in RP soil TN and TP concentrations and N:P ratios were smaller (Fig. 3b–c, f). and AD (Fig. 2a, e), while P concentration was larger (Fig. 2c), with Average SOC, soil TN, and TP concentrations were larger in RPAD than in similar findings in RPAS compared to RP and AS. Litter N:P ratio was RP and AD, and in RPAS than in RP and AS (Fig. 3a–c). In addition, the measured in both RPAD and RPAS between their corresponding mono- average SOC concentration, soil C:N, and C:P ratios was higher in RPAD cultures (Fig. 2f). Litter N and P concentrations and N:P ratio was higher than in RPAS (Fig. 3a, d–e), while shallow soil TN and TP concentrations in RPAD than in RPAS (Fig. 2b, c, f), while C concentration, C:N, and C:P and N:P ratio were smaller (Fig. 3b–c, f). ratios were smaller (Fig. 2a, d, e). The interaction between tree species Afforestation pattern and soil layer had significant effects on the SOC, and afforestation pattern had a significant effect on the litter C:N:P soil TN, and TP concentrations and C:N ratio (P < 0.001), though their stoichiometry (P < 0.01, Table S2). Tree species, afforestation pattern, interactions had inconsistent effects (Table S3). The average SOC and soil and their interaction had significant effects on the litter N and P con- TN concentrations and N:P ratio were larger in RPAD than in RP and AD, centrations, C:N, and C:P ratios (P < 0.01, Table S2). as well as being larger in RPAS compared to RP and AS (Fig. 3a–b, f). Although soil nutrient “surface aggregation”, soil C:N:P stoichiometry Afforestation pattern had a significant effect on the average SOC and soil decreased with soil depth (Fig. 3). Under the topsoil, soil nutrient con- TN concentrations and N:P ratio (P < 0.01, Table S4). centrations were larger in RPAD than in RP and AD, and in RPAS than in RP and AS (Fig. 3a–c); soil C:N ratios were smaller in RPAD than in RP 3.4. Relationship of C:N:P stoichiometry within the plant, litter, and soil and AD (Fig. 3d), while C:P and N:P ratios were larger than in RP and AD (Fig. 3e and f); C:N, C:P and N:P ratios in RPAS were between RP and AS In all plants structures (i.e., leaf, branch, stem, fine root), N and P 5 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 2. Litter C, N, and P concentration (a, b, c) and stoichiometric ratio (d, e, f) in mixed-species and monoculture plantations. Data are means  SE; different uppercase letters above the bars indicate significant differences among different plantations. concentrations were positively correlated (P < 0.05, Fig. 4c), in addition observed between stem and soil N:P ratio (P < 0.01, Fig. 5c and d) in to C:N and C:P ratios (P < 0.05, Fig. 4d). In litter and average soil, C and RPAD and RPAS, plant (leaf, branch, and root) and litter N:P ratio (P < N concentrations were positively correlated (P < 0.01, Fig. 4a), as well as 0.01, Fig. 5c and d) in RP, litter C:P and C:N ratios (P < 0.001, Fig. 5c and C:P and N:P ratios (P < 0.01, Fig. 4f). d) in AD, and plant (leaf, branch, and root) and soil C:N and C:P ratios (P A positive correlation was observed between litter N and P concen- < 0.05, Fig. 5c and d) in AS. trations (P < 0.05, Fig. 5a and b), as well as between SOC and soil TN concentrations (P < 0.05, Fig. 5a and b), in RPAD and RPAS. Positive 3.5. Effect of litter and soil on the plant C:N:P stoichiometry correlations were also found between plant (leaf, root) and soil P con- centrations (P < 0.01, Fig. 5a and b) in RP, stem N and P concentrations Litter N concentration exhibited a positive correlation with leaf, (P < 0.001, Fig. 5a and b), leaf and litter C concentrations (P < 0.001, branch, and root N concentration (P < 0.01, Fig. 6b) in both mixed- Fig. 5a and b) in AD, and plant (stem, root) and soil C concentrations (P species and monoculture plantations. Litter C:N and N:P ratios showed < 0.01, Fig. 5a and b) in AS. Similarly, positive correlations were a positive correlation with root C:N and N:P ratios (P < 0.05, Fig. 6d, f), 6 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 3. The vertical distribution and the average soil C, N, and P concentration (a, b, c) and stoichiometric ratio (d, e, f) in mixed-species and monoculture plantations. Data are means  SE; different uppercase letters above the bars indicate significant differences among different soil layers in the same plantation; different lowercase letters above the bars indicate significant differences among different plantations at the same soil layer. while litter C concentration displayed a positive correlation with leaf C stoichiometry, as well as with stem and root stoichiometric ratios, while concentration (P < 0.01, Fig. 6a). Furthermore, litter C and P concen- soil exhibited the strongest association with stem and root nutrient trations exhibited negative correlations with branch C concentration (P contents (Table 5). < 0.01, Fig. 6a) and stem P concentration (P < 0.01, Fig. 6c), respec- tively. Litter C:P ratio was negatively related to stem C:P ratio (P < 0.01, 4. Discussion Fig. 6e). Soil TP concentration showed a positive correlation with leaf and root P concentration (P < 0.01, Fig. 7b), whereas soil C:P ratio dis- 4.1. Comparison of C:N:P stoichiometry in plant, litter, and soil among played a positive relationship with leaf and root C:P ratio (P < 0.05, mixed-species and monoculture plantations Fig. 7d). Furthermore, soil TN concentration and C:N ratio exhibited negative correlations with stem N concentration (P < 0.05, Fig. 7a) and 4.1.1. Comparison of plant C:N:P stoichiometry among mixed-species and C:N ratio (P < 0.01, Fig. 7c). Variance partitioning analysis revealed that monoculture plantations litter demonstrated strong associations with leaf and branch C:N:P Wright et al. (2004) reported that tree species allocated the most 7 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 4. Relationships between C and N concentrations (a), C and P concentrations (b), N and P concentrations (c), C:N and C:P ratios (d), C:N and N:P ratios (e), and C:P and N:P ratios (f) in the plant (leaf, branch, stem, fine root), litter, and average soil of different plantations. nitrogen and phosphorus to their leaves, which is consistent with our Leaf N:P ratio is often used as an indicator to quantify the limiting results (Table 3). This may be because fast-growing tissues, such as the nutrient in soil (Güsewell, 2004; Han et al., 2013). In our study, the leaf, require more nitrogen and phosphorus for protein synthesis, growth of R. pseudoacacia in different plantations was limited by soil P resulting in lower C:N and C:P ratios (Table 4) (Yuan et al., 2011), and (Table 4), which is consistent with previous research (Cao and Chen, leaves must sustain all photosynthesis production to provide sources for 2017; Wu et al., 2021a; Jiao et al., 2022). Zhao et al. (1995) reported that all plant growth (Wang et al., 2015). Moreover, tree species are affected leguminous plants need to consume a large amount of phosphorus in the by their respective tissue structures and functional differentiation, lead- nodulation process, and their roots secrete organic acids to activate ing to unique nutrient requirements (Sardans et al., 2017). Consistent insoluble phosphorus, thereby promoting plant uptake of phosphorus with hypothesis 1, higher N concentrations were observed in leaves of from the soil. Besides, mixed stands of A. davidiana in RPAD was no A. davidiana in RPAD than A. sibirica in RPAS, and in R. pseudoacacia longer limited by soil N, while A. sibirica in RPAS was still limited by soil growing in RPAD than growing in RPAS (Table 3). A plausible explana- N (Table 4). Therefore, the ability of N2-fixing bacteria to alleviate ni- tion is that there is a segregation of species niche leading to differences in trogen limitation of symbiotic species may depend on the nitrogen re- dynamic nutrients use efficiencies and a corresponding dilution effect in quirements of the latter (Reich et al., 1992; Forrester, 2014; Yin et al., leaves (Bauhus et al., 2004). 2021). 8 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 5. Relationships among C, N, and P concentrations (a, b) and stoichiometric ratios (c, d) in the plant-litter-soil system of different plantations. 4.1.2. Comparison of litter C:N:P stoichiometry among mixed-species and Compared to corresponding monocultures, mixes of R. pseudoacacia with monoculture plantations A. davidiana, or A. sibirica, were found to alleviate N-limitation during Litter plays a major role in nutrient cycling between plants and soil, litter decomposition (Fig. 2d), indicating that mixes of N2-fixing and where organic matter is broken down into small inorganic molecules by symbiotic tree species can improve nitrogen retranslocation efficiency microbes. Lower C:N and C:P ratios were beneficial to microbial activity, (Townsend et al., 2007). In addition, the increased of litter decomposi- promoting faster rates of litter decomposition and nutrient release (Sar- tion rate was contributed to the N transfer rate (Britton et al., 2018). iyildiz and Anderson, 2003; Manzoni et al., 2010). Based on this infor- mation, our data suggest that mixed stands of A. davidiana with 4.1.3. Comparison of soil C:N:P stoichiometry among mixed-species and R. pseudoacacia have increased litter decomposition rates compared with monoculture plantations corresponding monocultures (Fig. 2d and e) (Farooq et al., 2022). This is Species diversity of plantation can influence the released of litter consistent with the observation that shallow soil nutrient concentrations products and root exudates into soil (Chapman and Newman, 2010; in mixed-species plantations were higher than in monocultures (Lü et al., Zhang et al., 2022). Moreover, in mixed stands, different species habit- 2012). However, the values of litter C:N ratio in mixed stands of ually do not exploit exactly the same soil layers and thus this makes A. sibirica and R. pseudoacacia were between the litter C:N ratio of their possible a better exploitation of soil resources and at some extent avoid respective monocultures (Fig. 2d), suggests a relatively stable microbial direct competition (Forrester et al., 2006; Khanna et al., 2008). Besides, abundance and diversity. Consistent with previous studies, senesced plantations with complex canopies structure are conductive to inter- leaves with greater lignification from A. sibirica in RPAS are difficult to cepting rainfall, resulting in reduced soil nutrients carried by spatter break down for microorganisms (Townsend et al., 2007; Laclau et al., erosion and leaching (Eviner and Chapin, 2002). For these reasons, 2010). average soil TN and TP concentrations were larger in RPAS than that in The decomposition of litter is limited by nutrients, which in the case RPAD, and larger in mixed-species plantations than in monocultures of nitrogen and phosphorus can be reflected by the N:P ratio (Güsewell (Fig. 3b and c) (Lü et al., 2012). Whereas similar litter P concentrations in and Verhoeven, 2006). The thresholds of litter N- and P-limitation occur mixed-species plantations indicate that increased shallow soil TP con- at 22 and 25 g⋅kg1, respectively (Güsewell and Freeman, 2005). centrations were not just caused by litter (Fig. 2c, Fig. 3c). Different 9 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 6. Relationships between plant (leaf, branch, stem, fine root) and litter in C, N, and P concentrations (a, b, c) and stoichiometric ratio (d, e, f) of different plantations. distributions of fine root systems in mixed plantations provide an nitrogen and phosphorus mineralization (Fig. 3b and c), which is bene- enabling environment for rhizobium activities (Singh et al., 2012). ficial to alleviate plants growth limited by soil nitrogen and phosphorus. Higher nitrogen availability frequently translates in higher Zhou et al. (2019) reported that soil C:P ratio gradually decreased with plant-microbes capacity to mobilizate and uptake other nutrients such as deepening soil, consistent with our results. This is because SOC con- phosphorus (Sardans and Pe~ nuelas, 2012). Thus, mixing R. pseudoacacia centration was declined gradually in different plantations, while soil TP with A. sibirica can improve N-cycling and the availability of soil phos- concentration was relatively stable across the soil profile (Fig. 3a, c) (Qiu phorus (Forrester et al., 2006). et al., 2010; Yang et al., 2018; Pang et al., 2021). N:P ratio can reflect the The soil C:N and C:P ratios can indicate the soil nitrogen and phos- status of soil nitrogen saturation and determine the threshold of nutrient phorus mineralization capacity, and a lower ratio demonstrates higher limitation (Tessier and Raynal, 2003; Cui et al., 2021). Here, our data net nitrogen and phosphorus mineralization (Bui and Henderson, 2013; suggest that mixtures of R. pseudoacacia with A. sibirica could alleviate Marty et al., 2017). Compared with RPAD, RPAS had the higher net N-limitation issues (Fig. 3f) (Zhu et al., 2013). 10 S. Lu et al. Forest Ecosystems 10 (2023) 100123 Fig. 7. Relationships between plant (leaf, branch, stem, fine root) and average soil in N and P concentrations (a, b) and stoichiometric ratio (c, d) of different plantations. No significant relationship was found between plant (leaf, branch, stem, fine root) and average soil in C concentrations and N:P ratio of different plantations. Table 5 Litter and soil C:N:P stoichiometry compared to plant (leaf, branch, stem, fine root) C:N:P stoichiometry. Fraction (%) Leaf C:N:P stoichiometry Branch C:N:P stoichiometry Stem C:N:P stoichiometry Root C:N:P stoichiometry Concentrations Ratios Concentrations Ratios Concentrations Ratios Concentrations Ratios A 53.9 49.5 65.3 34.5 36.2 35.3 37.5 50.0 B 19.0 10.1 14.6 15.1 47.4 9.5 46.6 18.8 C 20.0 19.0 5.1 16.8 16.5 29.8 8.0 20.6 Notes: A and B represents the separate explained of litter and soil, respectively; C represent the combined explained of litter and soil. 4.2. Relationships between C:N:P stoichiometry in plant, litter, and soil of rates and initial nitrogen and phosphorus concentrations (Wang et al., the mixed-species and monoculture plantations 2008), annual litter production was higher in mixed plantations than in monocultures (Farooq et al., 2022). Therefore, both N- and P-cycling Consistent with hypothesis 2, positive correlations were observed rates were faster in mixed-species plantations than in their respective between N and P concentrations and between C:N and C:P ratios in plant monocultures, and the mixed-species plantations had more active structures (i.e., leaf, branch, stem, fine root) (Fig. 4c and d), indicating biogeochemical cycles (Forrester et al., 2006). Furthermore, litter was that plants were able to use the increased nitrogen to allocate more re- positively correlated with the soil TN concentrations in mixed-species sources to phosphorus extraction from organic matter and promote soil P- plantations (P < 0.05, Fig. 5a and b), while similar results were not cycling (Reich et al., 1992). Additionally, stem C:P ratio was positively found between litter and soil TP concentrations (P > 0.05, Fig. 5a and b). correlated with the soil N:P ratio in RPAS, whereas similar results were This is inconsistent with previous studies in which 90 % of the nitrogen not found in monocultures (Fig. 4f). This indicated that mixed-species and phosphorus elements released from litter into the soil were taken up afforestation regulated the effects of soil TN and TP on stem phos- by plants (Chapin et al., 2011). Wang et al. (2008) and Farooq et al. phorus use efficiency (Yin et al., 2021), which was consistent with pre- (2022) explained that nitrogen returned to the soil by litter accounted for vious studies (Yang et al., 2018; Wu et al., 2021b; Jiao et al., 2022). 80 % of the total nutrients returning to the soil, while only 2 % of the total In addition to a positive correlation between litter decomposition nutrients returned through the litter was phosphorus. 11 S. Lu et al. Forest Ecosystems 10 (2023) 100123 4.3. Effect of litter and soil on plant C:N:P stoichiometry among mixed- Funding species and monoculture plantations This study was funded by the National Nature Science Foundation of Previous studies have demonstrated that leaf-to-litter conversion oc- China (No. 41771556). JP and JS were funded by the Spanish Govern- curs rapidly, and branches contain increased carbon reserves (Pang et al., ment Grants PID2020-115770RB-I00 and TED2021-132627B–I00 fun- 2021), which aligns with our finds (Fig. 6a, Table 5). Additionally, the ded by the Spanish MCIN, AEI/10.13039/501100011033 and the observed relationships among plant organs (leaf, branch, and root) and European Union NextGenerationEU/PRTR, the Catalan government litter N concentrations (Fig. 6b) and C:N ratios (Fig. 6d) strongly suggest grant SGR2021-1333 and the Fundaci on Ram on Areces grant that plants with high nitrogen requirements promote the rapid decom- CIVP20A6621. position of litter (Bradford et al., 2016). This implies a coupling strategy of “nutrient demand-supply” (Guo et al., 2021). However, we only found Authors’ contributions negative correlations between litter and stem P concentration (P < 0.01, Fig. 6c) and C:P ratio (P < 0.01, Fig. 6e). Thus, after phosphorus is YMC and SBL conceived and designed the study; SBL conducted the transported from the stem to the leaf, its transfer to other organs becomes experiment; SBL, YMC, JS, and JP analyzed the results, wrote and edited challenging. N:P ratios demonstrated a stronger relationship between the manuscript. litter and roots compared to leaves and branches (P < 0.01, Fig. 6f; Table 5), further suggesting that most litter decomposition products are Declaration of competing interest absorbed by roots, while only a portion of them is subsequently distrib- uted to branches and leaves, consistent with previous studies (Chapin The authors declare that they have no known competing financial et al., 2011). interests or personal relationships that could have appeared to influence Consistent with hypothesis 3, we only observed negative correlations the work reported in this paper. of N concentration and C:N ratio between stems and soil (P < 0.05, Fig. 7a–c), indicating that the stem is the plant organ more directly linked Acknowledgements to soil TN concentrations (Table 5). However, carbon and nitrogen in leaf, branch, and root can also originate from sources other than direct Not applicable. uptake from the soil, such as photosynthetic carbon fixation, and bio- logical nitrogen fixation (Mendez and Karlsson, 2005). Both leaves and Appendix A. Supplementary data roots are highly active plant organs, and their physiological traits are strongly associated with soil phosphorus (Ren et al., 2016), which is Supplementary data to this article can be found online at https://do consistent with our findings (Fig. 7b). This suggests that soil TP con- i.org/10.1016/j.fecs.2023.100123. centrations play a crucial role in nutrient reallocation within leaves and roots over long-term processes. Therefore, all the results indicate that the References majority of phosphorus released into the soil from litter is absorbed by the roots and subsequently transferred to the leaves. Bao, S.D., 2000. Soil and Agriculture Chemistry Analysis, third ed. 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