Direct and legacy effects of plant-traits control litter decomposition in a deciduous oak forest in Mexico

Field experiment

Brown color polyester mesh (1 mm) bags (10 × 10 cm) were used for the field decomposition experiment. The mesh size of 1 mm was chosen because it avoids losing small leaf litter debris but allows the activities of the aerobic microbial community and meso- and micro-fauna (Nguye Tu et al., 2011), which play an important role in the initial fragmentation of litter (Gessner et al., 2010). These bags were filled with 11 g of oven-dried litterfall with the following arrangement: 30 bags with Q. castanea litterfall (QcL), 30 bags with Q. deserticola litterfall (QdL) and 30 bags with a mixture of both species litterfall (QxL) in the same proportion (5.5 g of Q. castanea and 5.5 g of Q. deserticola litterfall). In June 2014, two litterbags of each litterfall type (QcL, QdL and QxL) were randomly located above the litter and around the stem of each of the five selected trees, distributed along the main slope, in each species condition plot (hereafter plots are referred to as sites): Q. castanea site (QcS), Q. deserticola site (QdS) and the species mixture site (QxS). Therefore, the field design is a complete factorial 3 × 3 (site and litterfall conditions). One litterbag for each treatment per tree was harvested at 30 and 270 days after the bags were placed (five bags for each treatment). The comparison of the two dates allows us to determine the decomposition effect on early and late decomposition stages, where the labile and recalcitrant molecules proportion changes over time. The means (± standard deviation) of DBH for trees in each condition were Qc: 52.9 ± 11.7 cm, Qx: 48.9 ± 4.9 cm and Qd: 63.9 ± 11.4 cm.

In the collection dates, the content of each bag was carefully removed, fresh field weighed, and subsequently divided into two subsamples. The first one was stored in hermetic bags in the dark at 4 °C until laboratory analysis. The second subsample was dried to constant weight at 70 °C for 72 h to calculate the water content. Then, the sample was milled in a ball mill at 350 RPM for three min and stored in sealed bags until chemical analysis.

Remaining mass and decomposition rate

Initial and remaining samples were combusted in a muffle furnace at 650 °C to determine inorganic particles to correct data on an ash-free basis. The litter decomposition rate was calculated using the simple exponential model: MR = Mi e^−kt; where MR is the percentage of the remaining mass at 270 days, Mi is the initial mass percentage, k is the decomposition rate and t is the decomposition time in field conditions (Olsen, 1963).

¹³C Nuclear Magnetic Resonance (¹³C NMR) spectroscopy

The ¹³C Nuclear Magnetic Resonance spectroscopy is a non-destructive analysis that improves the identification of the molecular composition from organic residues; it is useful tool for determination of the molecular composition of litterfall and decomposed litter. To characterize the chemical composition of litterfall and decomposed litter, the analysis of Cross Polarized Magic-Angle Spin ¹³C NMR in solid state was performed in samples previous to the field experiment (initial) and in samples at the end of the field experiment (remaining). The ¹³C NMR data were obtained at 298 K in a Varian Inova-750 17.6 T (operated at 750 MHz frequency proton), under the conditions described in Chavez-Vergara et al. (2014). The spectrogram obtained was processed with the program MestreNova V. 6 (Mestrelab Research Inc.).

For integration, the spectrogram was divided into four major regions representing different chemical environments of ¹³C nucleus according to position of relaxation signal in parts per million of chemical shift (ppm): C Alkyl (0–45  ppm), O-alkyl C (45–110 ppm), aromatic C (110–160 ppm) carbonyl and C (160–220 ppm). For more detailed analysis, spectra were divided according to Leifeld & Kögel-Knabner (2005) as: (I) 10–45 ppm C alkyl: methyl groups, methylene groups on rings and aromatic chains. (II) 45–110 ppm C O-alkyl: methoxy groups and C6 in some polysaccharides (45–60 ppm); C2–C5 hexoses C of some amino acids, aliphatic alcohols and fractions of lignin structure (60–90 ppm); Carbohydrate anomeric C, C2–C6 syringyl unit of lignin (90–110). III) 110–160 ppm aromatic C: and CC and CH carbon C2 guaiacil, C6 lignin (110–140 ppm, aryl C); COR aromatic or CNR (140–160 ppm, phenolic C) groups. IV) carboxyl 160–220 ppm C: carboxyl C, C carbonyl and C amide.

We also examined indexes associated with the decomposability of organic matter based on integrated specific regions: alkyl: O-alkyl ratio (A: OA), O-alkyl: aromatic ratio (OA: Ar), aromaticity (Ai), hydrophobicity (HB: HI) and characterization of lignin relations based on subunits specific regions such as syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H) as lignin relations S:G, S:H and G:H (Almendros et al., 2000; Spaccini et al., 2006; Talbot et al., 2012; Bonanomi et al., 2013; Chavez-Vergara et al., 2014).

Differential Scanning Calorimetry (DSC) and Thermogravimetry (TGA)

The Differential Scanning Calorimetry (DSC) and Thermogravimetry (TG) is a thermal analysis suitable for determination of organic matter stability (Angehrn-Bettinazzi, Lüscher & Hertz, 1988). This method quantifies the energy release during different combustion temperatures of samples, like the energy required for biological oxidation of organic molecules (Rovira et al., 2008). Therefore, the thermograms can quantify the proportion of labile, recalcitrant and extra-recalcitrant compounds in the organic samples (Barros, Salgado & Feijóo, 2007). The characterization of thermal properties of litterfall and decomposed litter was done by differential scanning calorimetry and thermogravimetric analysis (DSC-TGA, Mettler-Toledo International Inc.). The analysis was performed with 4 mg of powdered oven-dried sample placed in an aluminum pan in an atmosphere of dry air (flow rate, 50 ml min¹) and the scan rate was 10 °C min⁻¹. The temperature range used was 50 to 600 °C. An indium sample (melting point: 156.6 °C) was used to calibrate the calorimeter. All samples were analyzed in triplicate.

The combustion heat release (Q, J g⁻¹) was determined by integrating the DSC curves (W g⁻¹) on the exothermic region (150–600 °C). Data recorded at temperatures <150 °C were discarded because they are associated with the loss of mass and energy release during moisture loss. The Q value was divided by the mass loss in each measurement (Q′, J mg⁻¹ MO, Rovira et al., 2008).

Areas under the DSC curve were divided in three groups, representing different degrees of resistance to thermal oxidation (Dell’ Abate et al., 2002; Fernández et al., 2012): labile organic matter, comprising of carbohydrates and other aliphatic compounds (200–375 °C); recalcitrant organic matter such as lignin and/or polyphenols (375–475 °C); and extra-recalcitrant organic matter, such as polycondensed aromatic forms (475–550 °C). The heat release by combustion in each region was designated as Q₁, Q₂ and Q₃, respectively. Also, the temperature at which the maximum heat flow was detected during the combustion of organic matter (T₁, T₂ and T₃) and the temperature at which 50% of the total energy was released were recorded (T₅₀DSC).

Nutrient analysis

All forms of C were determined on a total carbon analyzer (UIC model CM5012, Chicago, IL, USA) by dry combustion and coulometric detection (Huffman, 1977), while forms of N were determined colorimetrically by the semi-Kjeldahl (Bremmer, 1996) method and the forms of P by molybdate colorimetric method after reduction with ascorbic acid (Murphy & Riley, 1962) in a Bran-Luebbe autoanalyzer (Autoanalyzer 3 Norderstedt, Germany) after acid digestion. The litterfall chemical analyses were performed from material collected from the respective traps (isolated Q. castanea, mixed species, and isolated Q. deserticola).

Soluble organic forms of C, N and P were extracted from 2 g of fresh material in deionized water, after stirring for 1 h and filtered through a Whatman# 42 filter and on a vacuum system through a 0.45 µm nitrocellulose membrane. The dissolved organic carbon (DOC) was determined by combustion coulometric detection (Huffman, 1977). Dissolved organic nitrogen (DON) and dissolved organic phosphorus (POD) were determined after acid digestion. The DON was calculated as the difference between the acid digested nitrogen and soluble NH${}_4^{+}$, as well as POD (acid digested P minus soluble inorganic P; Joergensen & Mueller, 1996).

Microbial biomass carbon (Cmic) and nitrogen (Nmic) were determined by direct extraction using chloroform fumigation (Brookes, Powlson & Jenkinson, 1984; Vance, Brookes & Jenkinson, 1987) from fresh samples. Two subsamples (2 g) were incubated at 30 °C for 24 h; one of the subsamples was maintained in chloroform atmosphere during incubation. Both samples were extracted in 0.5 M K₂SO₄ and percolated through # 42 Whatman filter paper and analyzed as DOC (as mentioned above) and removable N. The extractable N was quantified as total N after acid digestion (Brookes, Powlson & Jenkinson, 1984).

The microbial biomass P (Pmic) was determined by the fumigation-extraction method (Brookes, Powlson & Jenkinson, 1982) in fresh samples. A subsample of 0.5 g was fumigated for 24 h in a chloroform atmosphere and extracted with 30 mL of 0.5 M NaHCO₃ pH 8.5 for 30 min (Van Meeteren, Tietema & Westerveld, 2007). Extracts (fumigated and no-fumigated) were digested in a solution of sulfuric acid and persulfate ammonium according to Hedley sequential P fractionation (Tiessen & Moir, 2008) and quantified as orthophosphate, as described above. Cmic concentration, Nmic and Pmic were calculated from the difference between the fumigated and non-fumigated samples, then the concentration was corrected by applying the following factors: KeC 0.45 (Sparling et al., 1990), KeN 0.54 (Brookes, Powlson & Jenkinson, 1984; Joergensen & Mueller, 1996) and KeP 0.40, respectively (Brookes, Powlson & Jenkinson, 1982).

Enzyme activity

As a measurement of microbial activity related with the use of organic molecules, the enzymatic activities of β-1,4-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetyl-glucosaminidase (NAG), polyphenol oxidase (POX) and dehydrogenase (DHG) were determined in fresh material of each treatment for all samples collected. The determination of hydrolases (BG, CBH and NAG) was performed according to Chavez-Vergara et al. (2014) by colorimetric measurement of p-nitrophenol (pNP) in a spectrophotometer (Evolution 201, Thermo Scientific Inc., Waltham, MA, USA) at 420 nm liberated from specific substrates during incubation (2 h in oscillatory shaking at 30 °C) and reported in g-mol pNP litter⁻¹ h⁻¹.

POX activity was determined through oxidation of 2, 2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ATBS). One aliquot of the same extraction used for activity of hydrolases (Chavez-Vergara et al., 2014) was used, volume and time of preparation were the same as for hydrolases but in this case the result of the centrifugation was measured directly (without addition of NaOH and deionized water) on the same spectrophotometer described above by colorimetry at 460 nm and the result was reported as mol tyrosine g-litter⁻¹ h⁻¹, in this case the calibration curve is derived from tyrosine (tyr).

The dehydrogenase activity (DHG) was determined according to a modification of the method described by Alef (1995), which is based on the reduction of chloride of 2, 3, 5-triphenyltetrazolium (CTT) for the formation of triphenyltetrazolium formazan (TFF) in incubation at 30 °C for 24 h. To perform the assay 0.5 g of milled fresh material was weighed and placed in a conical tube with a capacity of 15 mL (the tube was covered with foil to keep out light) to which 1 mL of a solution CTT 1% in Tris buffer pH 7.6 was added. Blank samples were prepared only with the solution of CTT. Subsequently, all samples were placed and horizontally fixed in an incubator chamber; the incubation was for 24 h at 30 °C and 180 rpm. After incubation, 10 mL of acetone was added; the tube was stirred vigorously and allowed to react for 2 h in the dark at room temperature. Following, the supernatant was filtered through Whatman # 42 paper and measured at 546 nm by colorimetry. The measure of each sample was subtracted from the average value of blanks and adjusted by the equation described in Alef (1995). The results were expressed in g TPF g⁻¹d⁻¹.

The efficiency of enzymatic activity according to the concentration of a nutrient immobilized in the microbial biomass was calculated as specific enzyme activity (SEA) according to the following equation (Chavez-Vergara et al., 2014): (1)${\displaystyle \text{SEA}=A∕\text{Bmic}}$ where SEA is expressed in mol of pNP or mol of tyr released per milligram of nutrient in the microbial biomass per hour (mol mg⁻¹Bmic h⁻¹); A is the activity of any of the specific enzymes (BG, CBH, NAG, POX and DHG), and Bmic is the concentration of Cmic or Nmic in mg g⁻¹. The association of enzymes with nutrients in the microbial biomass is as follows: BG, CBH, POX and DHG are associated with Cmic, and NAG with Nmic.

¹³C CP MASS NMR characterization of litterfall samples

Figure 2 shows the spectra and Table 1 the integration of regions of ¹³C CPMAS NMR of litterfall and decomposed litter at 270 days in both species. In the original litterfall, the most prominent compounds were O-Alkyl, Aryl C and Alkyl C. Particularly, we observed that the region between 160 and 220  ppm, assigned to carboxyl/amide and carbonyl C groups, is dominated by a peak at ca. 173 ppm (Fig. 2) with similar relative C distribution for both species (Table 1). In the aromatic and phenolic region (160–110 ppm), the litterfall produced by both species showed well defined peaks at ca. 153 and 145 ppm, which revealed the presence of C3, C5 syringyl lignin and tannins, respectively. An incipient peak at ca. 131 in Q. deserticola and well-defined in Q. castanea may be related to unsubstituted and C-substituted phenyl carbon of lignin monomers of syringyl units (De Marco et al., 2012) affecting the S:G ratio between species (Table 1). In the O-alkyl region (45–110 ppm), the most prominent signal was at ca. 73 ppm and it was particularly associated to the simultaneous resonance of C-2, C-3 and C-5 of pyranose rings in cellulose and hemi-cellulose; and a second prominent peak was at ca. 105 ppm, traditionally associated to crystalline cellulose, and it can be associated to non-protonated carbon arising from tannins (Almendros et al., 2000).

A shoulder at 56 ppm, attributable to N-methoxyl C compounds in lignin, was relatively well resolved in litter samples of both species. Meanwhile, the C-6 position in the pyranose ring of cellulose produced the peak or shoulder at ca. 64 ppm better resolved in Q. castanea. In a similar way, that shoulder at 84 ppm may correspond to C-4 in cellulose (Almendros et al., 2000). The alkyl region (46–0 ppm) showed two well-defined peaks at 30 and 21 ppm. The peak at ca. 30 ppm is related to polyethylene carbons in lipids and lipid polymers such as cutine or suberine and the peak at ca. 21 ppm is frequently attributed to acetate groups in hemicellulose and/or short chain aliphatic structures. The most noticeable changes after 270 days of decomposition occur in the reduction of peak intensity in ca. 145 ppm associated to tannins and the increment of peaks at ca. 56 ppm (N-methoxyl) and two peaks in the alkyl C region (Fig. 2). These changes were most intense in the Q. deserticola litter.

Thermal characteristics of litterfall samples by differential scanning calorimetry (DSC)

The litterfall thermograms of both species showed a bimodal shape (Figs. 3A and 3B). The first peaks were situated at 347 °C and 352 °C, and the second prominent peaks were at 449 °C and 461 °C for Q. castanea and Q. deserticola, respectively (Table 2). Additionally, the Qc litterfall had a second peak at 424 °C in the Q2 region. However, the energy released from the recalcitrant region (Q2) was higher in the Q. castanea than in the Q. deserticola litterfall (Table 2). After 30 days of decomposition, the percentage of recalcitrant compounds (Q2) was higher in the Qc litter than in the Qd litter, while the Qd litter had the highest percentage of extra-recalcitrant compounds (Q3; Table 2; Figs. 3A and 3B). In the 270 days decomposed litter, the shape of the thermogram of Q. castanea remained as bimodal, while for Q. deserticola the third peak at the extra-recalcitrant region is well-defined and displaced to a higher temperature (503 °C; Fig. 3). The decomposed litter at 270 days increased the energy released, compared to 30 days, in the Q1 region (Qc + 4% and Qd + 2%), the Q2 region showed a decrease in the litter of both species (Qc − 7% and Qd − 4%; Table 2) and the Q3 region increased for both species but more for Q. castanea (Qc + 63% and Qd + 15%; Table 2). The values of T50 were displaced to lower temperatures (Table 2) in both species at 270 days of decomposition in comparison to 30 days of decomposition.

In the one-way ANOVA of the thermal parameters at 30 days of decomposition, recalcitrant and extra-recalcitrant compounds were the highest and the lowest in QcL and QdL, respectively, without a significant effect of site (Table 3). Figure 4 shows the thermograms of litter at 30 days of decomposition of each Quercus species condition within each site. The QdL showed the highest peak in the labile region at the three sites, while the QcL showed the highest peak in the recalcitrant region also in the three sites. However, only the QdL showed a small peak in the extra-recalcitrant region, mainly at the Qd site.

Nutrient concentration and stoichiometric ratios in litterfall and decomposed litter

The concentration of C was only affected by decomposition time (F = 3.5; p < 0.001) reducing its value at 270 days of decomposition (Table 4). In contrast, the interaction between litter quality and sampling date was significant for concentrations of N and P (F = 5.2, p = 0.01 and F = 5.5, p = 0.009, respectively). The QdL had higher N concentration in all decomposition dates in comparison with QcL and QxL (Table 4). However, the QxL and the QdL had higher P concentration than QcL at 30 and 270 days of decomposition, but these values were similar in the three species litterfall (Table 4). These results suggest that the QxL and QdL had microbial P immobilization after 30 days of decomposition. Therefore, the C:N and C:P ratios were affected by litter quality and sampling date (F = 25, p = 0.001 and F = 8.6, p = 0.004, respectively); the QcL had the highest values and the 270 days of decomposition had the lowest values for the three species conditions (Table 4). However, the N:P ratio was only affected by decomposition time (F = 17, p < 0.001), where the highest and the lowest values were in the litterfall and at 30 days of decomposition, respectively (Table 4).

In contrast, the dissolved organic carbon and nitrogen (DOC and DON, respectively) concentrations were affected by the interaction of litter quality and sampling date (F = 18, p < 0.001 and F = 21, p < 0.001, respectively). The QdL had higher DOC concentration than the QcL in the two decomposition dates (Table 4), although the QxL had a higher DOC reduction at 270 days of decomposition than the QcL (64% and 30%, respectively). In contrast, the QcL had lower DON than the QxL and QdL in the litterfall and the litter at 270 days of decomposition, but the three litters had no different DON values at 30 days of decomposition (Table 4). Additionally, the QdL showed a reduction of DON concentration only at 270 days of decomposition, while the litter of the two other species conditions had increments or no changes in relation to their litterfall (QdL and QxL, respectively). The DOP concentration was affected by both litter quality and sampling date (F = 18, p = 0.002 and F = 26, p < 0.00001, respectively); the QdL and QcL had the highest and lowest DOP concentration, respectively, and the highest and the lowest DOP values were at 30 and 270 decomposition days, respectively (Table 4).

Similarly, the dissolved inorganic forms of N (NH${}_4^{+}$ and NO${}_3^{-}$) were also affected by the interaction between litter quality and sampling date (F = 66, p < 0.001 and F = 9, p = 0.002, respectively). Both dissolved NH₄ and NO₃ concentrations were highest in the Qd and lowest in the Qc in the litterfall, but this pattern changed at the 270 days of the experiment for NH${}_4^{+}$ concentration (QcL>QdL = QxL; Table 4) and for NO${}_3^{-}$ concentration (Qd>QxL>QcL; Table 4). The dissolved inorganic P form (DiP) was affected by the main factors (F = 66, p < 0.001 and F = 26, p < 0.001 for litter quality and sampling date, respectively); the QdL and QcL had the highest and lowest DiP values, respectively, and the lowest DiP values were at 30 days of decomposition (Table 4).