Bleaching of leaf litter accelerates the decomposition of recalcitrant components and mobilization of nitrogen in a subtropical forest

Study site

The present study was conducted in evergreen broad-leaved subtropical forests in the northern part of Okinawa Island, south-western Japan. Samples were collected in a secondary forest within Yona Experimental Forest of University of the Ryukyus (26°9′ N, 128°5′ E, ca 250–330 m a.s.l.). The mean annual temperature was 20.7 °C and the annual precipitation was 2487 mm. The topography is hilly and dissected. The bedrock is composed of sandstone and slate, and yellow soil has developed. The forest stand was dominated by Castanopsis sieboldii and Schima wallichii ssp. liuliuensis with a maximum height of 20 m²⁸.

Litterbag experiment

Decomposition of leaf litter of six tree species (C. sieboldii, S. wallichii, Daphniphyllum teijsmannii, Persea thunbergii, Distylium racemosum, and Camellia japonica) was studied using a litterbag method, according to the procedure detailed previously¹³. These six tree species are dominant components of the forest canopy in the study site²⁸. In short, a study plot of 50 m × 10 m (500 m²) was laid out in Yona Experimental Forest and was divided into 125 grids of 2 × 2 m. Freshly fallen leaves of six tree species were collected from the soil surface in March 2008. The leaves were dried in an oven at 40 °C for 1 week to a constant mass. Leaf litter (4.00 g) was placed in litterbags (24 × 18 cm) made of nylon with a mesh size of approximately 2 mm and incubated within the 500 m² study plot for 18 months from April 2008 to October 2009. Nine litterbags per tree species were retrieved at 3, 6, 9, 12, 15, and 18 months after initiation of the experiment and used for measurement of the remaining mass of whole leaf litter¹³. In the present study, the bleached leaf area and leaf mass per area (LMA) and chemical compositions of bleached and nonbleached portions were then measured as described below. The LMA indicates the remaining mass of leaf tissues and represents the extent of decomposition in the bleached and nonbleached portions. Leaf litters of S. wallichii and D. teijsmannii collected at 12, 15 and 18 months of decomposition were too fragmented to measure bleached leaf area.

Measurement and chemical analyses

Leaves were pressed between layers of plywood and paper and oven-dried at 40 °C for 1 week. The leaves were photocopied, scanned, and measured for the total leaf area and the proportion of bleached area according to the method described previously²⁹. A 6-mm-diameter cork borer was then used to excise leaf disks, avoiding the primary vein, from the bleached area and surrounding nonbleached area of the same leaves collected for the first 9 months of decomposition. The disks were oven-dried again at 40 °C for 1 week and weighed to calculate LMA. The disks were combined to make 1 sample each of bleached and nonbleached leaf area for each tree species collected at each sampling occasion and used for chemical analyses as described below. Leaf disks could not be excised from leaves collected at 12, 15, and 18 months of decomposition because of fragmentation.

Litter materials were ground in a laboratory mill (0.5-mm screen). The amount of acid unhydrolyzable residue (AUR) and total carbohydrates (TCH) was estimated by means of gravimetry as acid-insoluble residue, using hot sulfuric acid digestion³⁰ and by a phenol–sulfuric acid method³¹. Total N content was measured by automatic gas chromatography (NC analyzer SUMIGRAPH NC-900, Sumitomo Chemical Co., Osaka, Japan). Details of the methods followed Osono¹³. The contents of AUR and TCH were expressed in g/g dry litter, and that of total N was in mg/g dry litter. The mass of these components per leaf area was calculated by multiplying the contents and LMA. The AUR fraction contains a mixture of organic compounds in various proportions, including condensed tannins, phenolic and carboxylic compounds, alkyl compounds such as cutins, and true lignin¹⁶. No data were available for total N content of the nonbleached portions of D. teijsmannii at 9 months of decomposition because of the small amount of sample.

To analyze the chemical composition of bleached leaf tissues more in detail and to compare it with that of nonbleached portions for multiple tree species, samples of bleached leaf litter were collected during fieldworks in March 2007 and in April 2011. These bleached leaf litter were separated into bleached and nonbleached litter samples to be used for measurement and chemical analyses (Table S1). Bleached leaf litter of 20 tree species was used for measurement of LMA, and the samples of 13 of the 20 tree species were further analyzed for the contents of AUR and TCH, as described above (Table S2). Samples were extracted with alcohol-benzene at room temperature (15–20 °C) to remove extractives (EXT; soluble polyphenols, hydrocarbons, and pigments) and to calculate the content of this fraction.

Solid-state Cross polarization (CP) magic angle spinning (MAS) ¹³C NMR spectra of bleached and nonbleached litter samples for 12 tree species were obtained with an Alpha 300 FT NMR system (JEOL, Tokyo) operating at 75.45 MHz under the following conditions³²: pulse repetition time of 3.1 s, CP contact time of 1 ms, sweep width of 35 kHz, acquisition time of 0.117 s, and MAS of 6 kHz. The finely powdered sample was tightly packed into a high-speed spinning NMR tube (rotor: zirconia, cap: KEL-F, 6-mm i.d., JEOL). Chemical shifts are quoted with respect to tetramethylsilane but were determined by referring to an external sample of adamantane (29.50 ppm). The ¹³C NMR spectra (Fig. S2) were divided into four chemical shift ranges, as follows: 0 to 45 ppm for alkyl-C (including major C of cutins and suberins), 45 to 110 ppm for O-alkyl-C (oxygen-substituted C in alcohols and ethers, including cellulose, hemicellulose, and other polysaccharides), 110 to 160 ppm for aromatic C (including mainly condensed tannins, hydrolyzable tannins, and lignin), and 160 to 190 ppm for carbonyl C (including carboxylic-C and carbonyl-C)³³. The relative area of these chemical shift regions was calculated for each spectrum as the percentage of total area by using computer software ALICE 2 for Windows (JEOL) (Table S3).

Nitrogen attached to leaf litter was determined by extraction and colorimetric analyses of the extractants for 13 tree species. Approximately 100 mg of bleached or nonbleached leaf litter was shaken with 10 ml of 2 M KCl in a 15-ml centrifuge tube on a shaker for 1 h. The suspension was centrifuged at 3000 rpm for 10 min and filtered with glass fiber filters (GF/F, Whatman). The total extractable nitrogen (TEN) in the extractants was measured by the alkali persulfate digestion method³⁴. Ammonium-nitrogen (NH₄⁺-N), nitrate-nitrogen (NO₃^–-N), and nitrite-nitrogen (NO₂^–-N) were determined colorimetrically³⁵ for the pre-digested samples. Extractable organic nitrogen (EON) was calculated subtracting these three forms of inorganic nitrogen from TEN (Table S4).

Statistical analyses

Linear relationships between LMA and decomposition time and between contents of AUR, TCH, and total N and accumulated mass loss of leaf tissue were examined separately for bleached and nonbleached portions according to the following equations:

Accumulated mass loss of leaf tissue of bleached and nonbleached portions after a given period was calculated as the loss of LMA relative to the initial LMA values, expressed as a percentage. Intercepts (a) and slopes (b) of regression equations were calculated for the linear relationships using least-square regression¹⁵. The slope of the regression Eq. (1) represented the decomposition constant³⁶. The slopes of the regression Eq. (2) describing AUR and N dynamics represented the N concentration increase rate and the lignin concentration increase rate, respectively¹⁵. A paired t-test was used to evaluate the difference between bleached and nonbleached portions in the slopes of regression equations for LMA, AUR, TCH, and N in decomposing leaf litter of 6 tree species and in LMA, contents of proximate organic chemical components, relative area of ¹³C NMR spectra, and contents of dissolved N in leaf litter of multiple tree species.

Source: Ecology - nature.com