The 1991. Some parcels have remained unafforested and covered

study was conducted in the Mazandaran wood and paper forest areas. The forest
ecosystem with the elevation scopes of 340-1200 m above sea level (a.s.l) is
located in the north of Iran (36° 27? N, 53° 04? E; Fig. 1). The average annual and monthly rainfall is about 726 mm
and <61 mm, respectively. The wet and dry seasons are September-March and April- August, respectively. Average daily temperature varies from 7.1 °C in January to 26.1 °C in August (Fig. 2). Nearly half a century ago, the area was dominated by natural forests with a number of native tree species, including oak (Quercus castaneifolia C. A. M), hornbeam (Carpinus betulus L.), ironwood (Parrotia persica C. A. Meyer). In the 1980s, due to extensive exploitation carried out by local residents, the Forests and Rangelands Organization of Iran (FROI) divided the area into several parcels, but in 1987, these parcels were clear-cut and then afforested by the FROI in different years since 1991. Some parcels have remained unafforested and covered by sparse herbaceous species such as Asperula odorata L., Euphorbia amygdaloides L., Hypericum androsaemum L., and Polystichum sp. The present investigation was performed on three old ages (15, 20, and 25 years) of afforested oak stands located between 350 and 360 m a.s.l, besides a barren land (without tree cover; WTC) located near the afforested stands and was selected as the control region. The selected parcels demonstrated broadly similar physiographic and climatic conditions as well as management applications (Kooch, et al. 2016). For simplification, hereafter the oak stand with 15, 20, and 25 years old were denoted as "OA1", "OA2" and "OA3", respectively.   Soil sampling and laboratory analysis           For each of the age categories of the afforested oak stand and WTC area three plots of about 1.5 ha were selected and transected. For each plot, five litter and soil samples (20 × 20 × 10 cm) were taken along two parallel transects, i.e., 120 litter and soil samples (3 plots at each site × 10 samples in each plot × 4 sites) were collected for laboratory analyses. The soil samples were stored in polyethylene bags at 4 °C until biological analysis. Litter was collected into bags, transported to the laboratory, washed gently for 30 s to remove mineral soil, and dried at 70 °C for 48 h. The dried samples were finely ground and analyzed.   Physicochemical properties analysis         Total C and nutrient contents in the litter samples were determined in quadruplicate using dry combustion with an elemental analyzer (Fisons EA1108, Milan, Italy) calibrated by the BBOT 2, 5-bis-(5-tert-butyl-benzoxazol-2-yl)-thiophen standard (Ther moQuest Italia s.p.a.) (Kooch, et al. 2012). Litter thickness was measured using a tape from the litter surface to top of the mineral soil (Dechoum, et al. 2015). The collected soils were air-dried, powdered and passed through a 2-mm sieve. Soil water content was measured following drying the samples in an oven at 105 °C for 24 h. To measure C and N contents in micro and macro-aggregates, the soil samples were wet-sieved under water by gently moving the sieve 3 cm vertically 50 times over period of 2 min through water contained in a shallow pan. The separated materials on the sieve were transferred to an aluminum container, dried at 60 °C in a forced-air oven and weighed (Cambardella and Elliott, 1992). Electrical conductivity (EC) and pH and of the soils were determined using an Orion Ionalyzer Model 901 in a 1:2.5 soil: water solution. The organic carbon and total N were determined according to the Walkley-Black (Allison, 1975) and semi Micro-Kjeldahl techniques (Bremner and Mulvaney, 1982), respectively.         C and N sequestration were calculated using the method described by Plaster's (1985). Available K, Ca, and Mg (following ammonium acetate extraction at pH 9) as well as P in the soils were determined using an atomic absorption spectrophotometer (Bower, et al. 1952) and a spectrophotometer (Homer and Pratt, 1961), respectively. Humic acid (HA) and fulvic acid (FA) contents in the soil samples were isolated and purified according to the method established by the International Humic Substances Society (IHSS) (Sparks and Bartels, 1996). To assess the urease activity, 200 mM urea was used as the substrate under standard condition (2 h at 37?C). Acid phosphatase activity was determined using 15 mM p-Nitrophenyl phosphate disodium (PNP) in a modified universal buffer (MUB) at pH 6.5 and incubated for 1 h at 37?C. Arylsulphatase activity was evaluated following incubation of the soils with p-Nitrophenyl sulphate (25 mM) for 1 h at 37?C and measurement of the quantity of p-Nitrophenol liberated during enzymatic hydrolysis by spectrophotometry. To measure the activity of invertase, 1.2% sucrose solution was used as substrate and incubated for 3 h at 50?C, and the released reducing sugars were quantified (Schinner and von Mersi, 1990).   Soil biological properties        Due to top soil sampling (10 cm deep) in this study, only superficial earthworm species, epigeic, was observed. The earthworms were collected by hand sorting, washed in water, and the wet weight was calculated with milligram precision. The removed earthworms were counted in the field, transferred to the laboratory, and oven dried at 60 °C for 24 h. Earthworm biomass was determined after drying (Kooch, 2012). Nematodes were extracted from 100 g soil sample by a modified cotton-wool filter method (Liang, et al. 2009). In addition, fine roots (< 2 mm diameter) were separated and dried at 70 °C to a constant mass (Neatrour et al. 2005).      Soil microbial analysis         Soil basal respiration (BR) was determined by incubating soil samples for 3 days at 25°C and finally measuring the amount of CO2 evolved (Alef, 1995). Substrate induced respiration (SIR) measurement was performed using glucose 1% as the substrate; after 72 h incubation the evolved CO2 trapped in NaOH was measured by HCl titration (Anderson and Domsch, 1990). Microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were measured by the fumigation-extraction method (Brookes, et al. 1985). Metabolic quotient (qCO2) was calculated as BR:MBC (Anderson and Domsch, 1990). Microbial entropy (MBC/C) (Insam and Domsch, 1988) and C availability index (BR/SIR) (Cheng, et al. 1996) were calculated based on the values of OC, BR, SIR and MBC. The Carbon availability index (CAI) for each soil was calculated as dividing BR by SIR (Cheng, et al. 1996). Carbon pool index (CPI) and liability index (LI) were calculated on the basis of changes in total carbon and in the proportion of labile C in the soil of the reference site (i.e., WTC) and in the soil of the afforested oak stands, respectively. Carbon management index (CMI) was derived from CPI and LI (CMI = C pool index (CPI) × lability index (LI) × 100 (Blair, et al. 1995). Particulate organic carbon (POC) was physically fractionated according to Cambardella and Elliot (1992). The concentration of NH4+ and NO3- in the soil samples were measured using a colorimetric method. Briefly, these substances were extracted with a 2 M KCl solution (soil: solution, 1:5) and filtered through a 0.45 µm filter. The absorbance of NH4+ and NO3- in the extracted solutions was determined at 645 and 420 nm, respectively (Li, et al. 2014). N mineralization (Nmin) was measured according to Robertson's method (1999), under controlled conditions. Dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) were measured using a Shimadzu TOC-550A total organic carbon (TOC) analyzer; DON was calculated as the difference between the total dissolved nitrogen reading and the combined NH4+ and NO3- reading (Jones and Willett, 2006). Statistical analysis        Statistical analysis was performed using the SPSS software package version 20. The Kolmogorov- Smirnov test and levene's test were used to assess the normality of the variables and the equality of the variances, respectively. One-way analysis of variance (ANOVA) was used to compare the considered litter and soil properties among the afforested and unafforested oak stands. Duncan test was further applied to test for differences at the P = 0.05 level. Principal component analyses (PCA) was used to find out associations between multivariate variables. Multivariate correlation was used to identify relationship between the variables and principal components using PC-ORD (version 5.0).


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