Litter decomposition and humus characteristics in Canadian and German spruce ecosystems: information from tannin analysis and 13C CPMAS NMR

Soil Biology & Biochemistry 32 (2000) 779±792

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Litter decomposition and humus characteristics in Canadian and German spruce ecosystems: information from tannin analysis and 13 C CPMAS NMR Klaus Lorenz a,*, Caroline M. Preston b, Stephan Raspe a, Ian K. Morrison c, Karl Heinz Feger a, 1 a

Institut fuÈr Bodenkunde und WaldernaÈhrungslehre, UniversitaÈt Freiburg, Bertoldstr. 17, 79085 Freiburg, Germany b Paci®c Forestry Centre, Natural Resources Canada, 506 West Burnside Rd., Victoria, BC, Canada V8Z 1M5 c Great Lakes Forestry Centre, Natural Resources Canada, 1219 Queen St. E., Sault Ste. Marie, Ont., Canada P6A 5M7 Accepted 18 October 1999

Abstract In¯uences of litter and site characteristics were investigated during the decomposition of black spruce (Picea mariana (Mill.) B.S.P.) and Norway spruce (Picea abies (L.) Karst.) needle litter in litterbags in two black spruce sites in Canada (6 and 12 months) and two Norway spruce sites in Germany (6 and 10 months). Mass losses were greater for black spruce litter (mean 25.2%) than for Norway spruce (20.8%), despite lower quality of black spruce litter in terms of lower N (10.1 versus 17.1 mg gÿ1), higher C-to-N ratio (49.0 versus 30.3) and higher content of alkyl C (surface waxes and cutin), indicated by CPMAS 13C NMR spectroscopy. However, Norway spruce litter was higher in condensed tannins than black spruce (37.8 and 25.3 mg gÿ1, respectively). Tannins were lost rapidly from both species, especially in the ®rst 6 months, with losses in 10±12 months of 75± 89% of the fraction extractable in acetone/water and 40±70% of the residual fraction. Losses were greater in the German sites (mean 75.2%, 10 months, versus 68.4%, 12 months), which had earthworms present and higher temperature, precipitation and catalase activity, the latter being positively correlated with tannin loss. There was a much larger contrast in the organic layers; with the Canadian sites having lower C-to-N ratios and higher N concentrations (C-to-N, 20.3 and 29.7; N, 26.0 and 13.8 mg gÿ1 for Canadian and German sites, respectively). The 13C NMR spectra showed that they were poorly decomposed and unusually high in condensed tannins (consistent with chemical analysis of 28.7 and 37.6 mg gÿ1, Canada; and 3.5 and 5.0 mg gÿ1, Germany), with depletion of lignin structures. Di€erences in other inputs (bark, wood, roots, understorey vegetation) and in site properties (climate, decomposer community, earthworm activity) may be responsible for the considerable di€erences in humus properties, which would not be expected from di€erences in the chemical composition and short-term decomposition of needle litter. The tannin accumulation, lignin depletion and N sequestration in the black spruce sites may be related to accumulation of unavailable N and associated forest management problems in these ecosystems. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Condensed tannins;

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C CPMAS NMR; Litter decomposition; Black spruce; Norway spruce; Catalase; Forest ¯oor

* Corresponding author. Present address: Institut fuÈr Bodenkunde und Standortslehre, UniversitaÈt Hohenheim, Emil-Wol€-Str. 27, 70593 Stuttgart, Germany. Tel.: +49-711-459-4066; fax: +49-711459-4067. E-mail address: [email protected] (K. Lorenz). 1 Present address: Institut fuÈr Bodenkunde und Standortslehre, UniversitaÈt Hohenheim, Emil-Wol€-Str. 27, 70593 Stuttgart, Germany

1. Introduction After climate, decomposition of leaf litter is in¯uenced by substrate quality factors, primarily nutrient contents and organic composition (Moore et al., 1999). Consideration of the latter is usually based on the results of proximate analysis, especially the acid-sol-

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 2 0 1 - 1

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uble fraction (mainly re¯ecting cellulose and hemicellulose content) and the acid-insoluble residue (commonly referred to as lignin or Klason lignin ). However, most tree and shrub litters also contain considerable proportions of two other biopolymers, cutin and tannin (Northup et al., 1995a; Ayres et al., 1997; Preston et al., 1997; Preston, 1999). In addition to constituting a signi®cant fraction of litter mass, tannins may a€ect several aspects of ecosystem functioning. They can reduce the palatability and digestibility of plants to insects and herbivores (Schultz et al., 1992) and inhibit microbial activity and N availability, possibly because of their protein-binding properties (Horner et al., 1988; Kuiters, 1990; Howard and Howard, 1993; Schimel et al., 1996, 1998). Recent studies on N release from litter of many Mediterranean and tropical species have emphasized the need for incorporating a measure of polyphenol content or protein-binding into litter quality indices (Gallardo and Merino, 1993; Handayanto et al., 1997; Mafongoya et al., 1998). It has also been proposed that high tannin or polyphenolic content in plants can provide a mechanism to conserve N in a thick humus layer in nutrient-limited environments (Northup et al., 1995a,b). However, despite great interest in the role of condensed tannins in litter decomposition and ecosystem function, there have been few studies providing unequivocal identi®cation and quanti®cation of condensed tannins, or making full use of the information available from solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. In addition to litter quality and climate, decomposition in forest ecosystems is also in¯uenced by other

site factors including humus type, parent material and faunal activity (Cortez, 1998). We studied decomposition of needle litter from two species, black spruce (Picea mariana (Mill.) B.S.P.) and Norway spruce (Picea abies (L.) Karst.). The litterbag study was carried out at two black spruce sites in Canada (6 and 12 months) and two Norway spruce sites in Germany (6 and 10 months). This design allowed comparison of the e€ects of litter quality and site properties over two periods, although scheduling problems did not allow exact comparison of 1 year results. With increasing stand age, northern black spruce forests develop nutrient limitation, at the same time as N is increasingly sequestered in the forest ¯oor (Pastor et al., 1987; Smith et al., 1998). In this study, in addition to changes in litter mass, C and N, we characterized litter and forest ¯oor samples using speci®c analyses for condensed tannins, and solid-state 13C nuclear magnetic resonance spectroscopy with cross-polarization and magic-angle spinning (CPMAS NMR). 2. Materials and methods 2.1. Plant materials In Germany, litter decomposition was studied in two experimental areas, Schluchsee and Villingen, located at higher altitudes of the Black Forest (SW Germany) (Project ARINUS; Feger et al., 1990; Armbruster, 1998; Raspe et al., 1998). The Canadian study areas, Black Sturgeon and Lake Nipigon Forests, are located at lower altitudes in northern Ontario (Foster et al.,

Table 1 Site properties of the four study areas Canada

Latitude Longitude Elevation (m asl) Mean annual temperature (8C) Annual precipitation (mm) Bedrock Soil pro®le (FAO) Humus form O-layer pH (H2O) pH (CaCl2) Dominant tree species Stand age (yr) a b

Germany

Black Sturgeon

Lake Nipigon

Schluchsee

Villingen

49812'N 88843'W 300 0.2a 784a (43% snow) Precambrian red sandstone Ferro-humic podzol Mor

49826'N 87848'W 450 0.2a 784a (43% snow) Granite outcrop Ferro-humic podzol Mor

47849'N 886'E 1150±1250 4.5b 1910b (30% snow) `BaÈrhalde' granite Haplic podzol Mull/moder

48849 'N 8822 'E 870±945 6.3b 1330b (25% snow) Quartz-sandstone Dystric cambisol Mor

3.8 3.4 Black spruce 50

4.5 3.7 Black spruce 120

3.5 2.8 Norway spruce 50

3.4 2.7 Norway spruce 100

Climate data are 30-year normals from the nearby weather station at Beardmore, Ont. (49837'N, 87857'W; 305 m asl). Climate data are 8-year normals (Armbruster, 1998).

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

1986; Scaratt, 1996). Table 1 shows general features of the sites. 2.2. Sample collection and processing In the summer of 1995, brown needles of logged black spruce trees were collected at the Black Sturgeon Forest study area in Canada. Meanwhile, brown needles of logged Norway spruce trees were collected at the German site Schluchsee. After oven-drying (308C) and sterilizing (fumigation for 72 h with 240 g methyl bromide mÿ3), 10 g portions of needle litters were placed in polyester net litterbags (12  12 cm; 1 mm mesh). In November 1995, 40 litterbags were exposed in four blocks (1 m2) at each of the Schluchsee, Villingen, Black Sturgeon and Lake Nipigon sites. In spring and fall (after 6 and 10 months), 20 litterbags were collected at each German site. In Canada, after 6 months 20 litterbags were collected at each site and 12 litterbags after 12 months. Due to scheduling problems, incubation time at the German sites was shorter and in the fall a smaller number of litterbags was collected at each Canadian site. Litterbags were handsorted to remove debris and mesofauna. Litter aliquots from each bag were dried at 708C to constant mass and weighed for calculation of mass loss. For the proanthocyanidin assay and for chemical analysis, dried composite samples from each site and for each date of collection were ground in a Wiley mill to pass through a 0.85 mm sieve. In autumn 1995, samples of the organic layer were collected at all sites and were treated in a manner similar to the litter samples. Also, for each site and date of collection, ®eld-moist litter was pooled into a composite sample to measure microbial activity (for consistency, moisture was adjusted to 60% of water capacity). 2.3. C and N analysis Total C and N contents of litters and organic layers from the German sites were analyzed by ¯ash combustion using a Carlo Erba CNS Analyser Model NA 1500/AS 200. For samples from the Canadian sites, C and N contents were detected by automatic combustion using a Leco model CR12 Carbon Analyser and a LECO FP-228 N Analyser. 2.4. Proanthocyanidin (PA) assay The procedure was modi®ed slightly from those described by Preston et al. (1997) and Preston (1999). The reagent, prepared each day, was 5% concentrated HCl in n-butanol (v/v), with a total water content of 5% v/v and 200 mg lÿ1 of Fe2+ as FeSO4
7H2O. A standard solution (0.5 mg mlÿ1 in methanol) was prepared using a puri®ed condensed tannin from tips of

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balsam ®r (Abies balsamea (L.) Mill.) (Preston et al., 1997). Samples of litter and humus were analysed in two stages to determine extractable and residual tannins. For extraction, samples were weighed into centrifuge tubes using 25 mg for fresh litter and 50 mg for decomposed litter and organic layers. After adding 20 ml of acetone/water (70:30) (v/v), the tubes were shaken for 1.5 h. After centrifugation at 7000 rpm for 20 min, the extracts were decanted into 50 ml volumetric ¯asks followed by a second extraction and centrifugation. The extracts were combined and acetone/water added to 50 ml. The insoluble residue was air dried for analysis of residual tannins. For the assay, 2 ml aliquots of extract were added to screw-cap test tubes and dried at room temperature with a stream of air, because tests showed that ovendrying aqueous acetone extracts at 708C seriously degraded the response. This was not a problem with solutions of puri®ed tannin in methanol, for which aliquots of 0.05 to 0.25 mg were dried at 708C. The extraction residues were also transferred into screwcap test tubes for analysis. For hydrolysis, 5 ml of reagent was added to all samples (extracts, residues, standards, blank), which were brie¯y stirred with a vortex mixer, then heated in a water bath at 958C for 1 h. After cooling, solutions were transferred into disposable cuvettes. Tubes for residue analysis were centrifuged before transferring the solutions. Solutions were scanned from 430 to 750 nm and absorbances were determined at the peak maximum, at 555 2 2 nm, with allowance for sloping baseline. If necessary, samples were diluted with reagent to bring the absorbance into the usable range. Sample contents were determined from the calibration curve established for balsam ®r tannin. Total condensed tannin content was calculated as the sum of extractable and residue tannins. 2.5.

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C CPMAS NMR spectroscopy

Solid-state 13C NMR spectra of litter and organic layers with cross-polarization and magic-angle spinning (CPMAS NMR) were obtained using a Bruker MSL 300 spectrometer (Bruker Instruments, Karlsruhe, Germany) operating at 75.47 MHz. Dry, powdered samples were spun at 4.7 kHz in a 7 mm OD rotor. Spectra were acquired with 1 ms contact time, 2 s recycle time and 6000 scans and were processed using 30±40 Hz line-broadening and baseline correction. Chemical shifts are reported relative to tetramethylsilane (TMS) at 0 ppm, with the reference frequency set using adamantane. Dipolar dephased (DD) spectra were generated by inserting a delay period of 40±50 ms without 1H decoupling between the cross-polarization and acquisition portions of the CPMAS pulse sequence. All DD spectra were obtained

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Table 2 Mean values of mass loss (2standard deviation) during needle litter decomposition at the Canadian sites and the German sitesa Species

Site

Six-month …N ˆ 20† (%)

Ten-month …N ˆ 20† (%)

Twelve month …N ˆ 12† (%)

Black spruce

Schluchsee Villingen Black Sturgeon Lake Nipigon Schluchsee Villingen Black Sturgeon Lake Nipigon

16.222.1a 18.221.5b 16.222.9a 19.623.6b 10.323.0 13.021.7c 14.222.6c,d 15.823.5d

23.122.0 26.622.3 ND ND 19.524.3 22.522.4 ND ND

NDb ND 28.124.3 23.022.1 ND ND 21.624.4 19.422.5

Norway spruce

a b

Six-month means of mass loss with the same letter are not signi®cantly di€erent at P < 0.05 (Mann±Whitney-U-test). ND ˆ no determination.

using the TOSS sequence for Total Suppression of Spinning Sidebands. The NMR spectra of litters were divided into chemical shift regions as follows: 0±50 ppm, alkyl C; 50±60 ppm, methoxyl C; 60±93 ppm, O-alkyl C; 93±112 ppm, di-O-alkyl C and some aromatics; 112±140 ppm, aromatic C; 140±165 ppm, phenolic C; and 165±190 ppm, carboxyl C. The chemical shift regions for spectra of organic layers were as follows: 0±48 ppm, alkyl C; 48±90 ppm, methoxyl and O-alkyl C; 90±110 ppm, di-O-alkyl C; 110±137 ppm, aromatic C±C and C±H; 137±160 ppm, aromatic C±O and C±N; 160±187 ppm, carboxyl, amide and ester C; 187±210 ppm, aldehyde and ketone C. Areas of the chemical shift regions were determined after integration and expressed as percentages of total area (`relative intensity'). There are limitations in the quantitative reliability of CPMAS spectra, but it is appropriate to use them to compare intensity distributions among similar samples and to use DD spectra to point out structural features (Preston et al., 1994, 1997). 2.6. Determination of catalase activity For characterization of microbial activity during decomposition, we measured activity of the enzyme cata-

lase, which in litter originates predominantly from microorganisms. This activity is expressed by the percentage of O2 generated relative to the maximum volume that can be split from a given volume of H2O2 during 3 min (Beck, 1971). 2.7. Statistical analysis Results of 6-month mass loss were based on arithmetic means (2standard deviation) of 20 samples at the Canadian sites and 20 samples at the German sites. For statistical validity of the results the nonparametric Mann±Whitney-U-test (Sachs, 1978) was applied to compare two means. The signi®cance level of 95% was used. Comparison of mean values was performed with the statistical program package SPSS. Due to scheduling problems, total incubation time at the German sites was shorter (10 months compared to 12 months at the Canadian sites). For this reason, statistical analysis of mass loss was only done for the 6-month mass data. The 10 and 12month data are not directly comparable, but provide a guide to changes close to 1 year of decomposition. The tannin analyses were carried out on composite samples from each site and are interpreted qualitatively.

Table 3 Signi®cant di€erences in mean values of mass loss after decomposition at the Canadian and the German sites (Mann±Whitney-U-test; 0.001; 0.001 < P < 0.01; 0.01 < P < 0.05)

After 6 months Black spruce Norway spruce After 10 or 12 months Black spruce Norway spruce a

NS ˆ not significant.

Schluchsee Villingen

Black Sturgeon Lake Nipigon

Schluchsee Black Sturgeon







NS

NSa







NS





P<

Schluchsee Lake Nipigon

Villingen Black Sturgeon

Villingen Lake Nipigon







NS

NS 

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

Fig. 1. Tannin loss and catalase activity after decomposition of spruce needle litter (10 months in Germany, 12 months in Canada).

3. Results 3.1. Mass, chemical and biochemical changes Litter mass losses after 6 and 10 or 12 months of decomposition are shown in Table 2 and signi®cant di€erences after 6 months and after 10 or 12 months in Table 3. Mass losses after 6 months ranged from 10.3 to 19.6% and were higher for black spruce at all sites, with the greatest di€erence being at Schluchsee (10.3% for black spruce and 16.2% for Norway

783

spruce). This species e€ect persisted for the second sampling, when mass losses for black spruce were higher than those for Norway spruce litter within each site. Due to the di€erent ®eld exposure times, losses from Canadian versus German sites cannot be compared directly, but they are similar, especially for Norway spruce. Some signi®cant site e€ects were seen at both decomposition times. At 6 months, for black spruce, mass losses were lower (highly signi®cant, P < 0.001) at Schluchsee than at Villingen and Lake Nipigon, and signi®cantly (0.001 < P < 0.01) lower at Black Sturgeon than at Lake Nipigon. For Norway spruce, highly signi®cant di€erences in mass losses were found between Schluchsee and both Black Sturgeon and Lake Nipigon sites, and mass loss at Schluchsee was also signi®cantly lower than at Villingen. At 10 months, losses at Schluchsee remained lower than at Villingen for black spruce (highly signi®cant) and for Norway spruce (signi®cant). After 12 months, mass losses for Norway spruce did not di€er signi®cantly between the two Canadian sites. For black spruce, the di€erence between sites was only at the 0.01 < P < 0.05 level. For the German sites, mass losses at Schluchsee were always less than at Villingen for both species and sampling times. For the Canadian sites, however, site e€ects were more variable; mass losses for black spruce were larger after 6 months at Lake

Table 4 Tannin contents and tannin loss (% of initial) during decomposition of spruce needles and tannin contents of site organic layers Sample

Decomposition (months)

Black spruce

0 6 10, 12 0 6 10, 12

Norway spruce Organic layer Black spruce Norway spruce Organic layer Black spruce Norway spruce Organic layer a b

Six and 10 months. Six and 12 months.

0 6 10, 12 0 6 10, 12 0 6 10, 12 0 6 10, 12

Schluchseea Extract (mg gÿ1) 11.3 2.1 (81.0) 1.8 (84.0) 26.2 6.0 (77.1) 3.8 (85.5) 1.5 Residue (mg gÿ1) 14.0 5.9 (57.6) 5.2 (62.5) 11.6 8.7 (25.6) 6.7 (42.7) 2.0 Total (mg gÿ1) 25.3 8.0 (68.1) 7.0 (72.1) 37.8 14.7 (61.0) 10.4 (72.6) 3.5

Villingena

Black Sturgeonb

Lake Nipigonb

1.7 (84.7) 1.4 (87.2)

5.0 (55.3) 2.9 (74.5)

4.5 (60.0) 2.7 (76.0)

4.8 (81.7) 2.8 (89.3) 2.1

10.1 (61.5) 5.7 (78.2) 14.2

8.3 (68.3) 4.5 (82.8) 23.6

6.3 (54.8) 4.2 (69.8)

6.7 (52.3) 5.0 (64.6)

6.7 (51.8) 6.0 (57.2)

7.0 (40.2) 5.3 (54.7) 2.9

8.4 (28.2) 7.0 (40.2) 14.5

8.3 (29.1) 5.9 (49.6) 14.0

8.0 (68.1) 5.6 (77.6)

11.7 (53.6) 7.9 (69.0)

11.2 (55.5) 8.7 (65.5)

11.9 (68.6) 8.2 (78.5) 5.0

18.5 (51.1) 12.7 (66.4) 28.7

16.6 (56.2) 10.3 (72.7) 37.6

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Table 5 Carbon and N content and C-to-N ratio of spruce needle litter (decomposed 10 months in Germany, 12 months in Canada) and of organic layers Sample

Intact litter

Schluchsee

Black spruce litter Norway spruce litter Organic layer

519.1 515.7

516.5 515.4 342.9

Black spruce litter Norway spruce litter Organic layer

9.2 13.6

10.1 17.1 12.5

Black spruce litter Norway spruce litter Organic layer

56.3 38.1

51.1 30.1 27.4

C (mg gÿ1)

N (mg gÿ1)

Nipigon, but after 12 months, were greater at Black Sturgeon. Norway spruce showed the same trend, but the di€erences were not signi®cant. Table 4 summarizes data for contents of extractable, residual and total condensed tannins in organic layers, and in needle litters before and after two periods of decomposition. Norway spruce litter was higher in total and extractable condensed tannin than was litter from black spruce (total tannin, 37.8 and 25.3 mg gÿ1, respectively), although both had similar contents of residual tannin. For both species and for all sites, concentrations of both extractable and residual tannins decrease. For the extractable fraction, losses after the second sampling period were over 80% at the German sites and over 70% at the Canadian sites. Percentage losses of the residual tannin fraction were less, but also tended to be higher at the German sites, as was the loss of total tannins. Compared to the di€erences between Canadian versus German sites, species di€erences were small and inconsistent. Fig. 1 shows tannin loss (as percentage of original concentration) plotted against catalase activities determined at the end of the ®eld exposure periods. Values for catalase activity were higher at the German sites, with the maximum found for Norway spruce litter at Villingen (18.4% gÿ1). This site also had the highest mass loss for Norway spruce, although the range of mass loss for Norway spruce was not large. There was no signi®cant relationship between catalase activity and mass loss or between tannin loss and mass loss. There was, however, a strong positive correlation between catalase activity and tannin loss, with the greater losses of tannins and highest catalase activities generally observed at the German sites. However, the high tannin loss for Norway spruce at Lake Nipigon (72.7%) was also associated with the highest catalase activity at a Canadian site (9.2% gÿ1). Both species had similar total C contents in fresh lit-

C-to-N

Villingen

Black Sturgeon

Lake Nipigon

514.5 512.4 474.5

507.6 503.8 538.6

507.3 502.5 515.0

10.5 16.9 15.0

10.4 14.7 28.8

10.7 15.2 23.2

49.0 30.3 31.6

48.8 34.3 18.7

47.4 33.1 22.2

ter (Table 5) and showed only small decreases at both sites. In contrast, N contents increased during decomposition. The biggest increases were found for Norway spruce at the German sites while smaller changes were observed at all sites for black spruce. The latter had a lower N content (9.2 mg gÿ1) than Norway spruce (13.6 mg gÿ1) and a higher C-to-N ratio. During decomposition, C-to-N ratio decreased for both species and all sites, but always remained higher for black spruce litter than for Norway spruce. There were large di€erences between the organic layers of the German and Canadian sites. The latter receive black spruce needle litter of C-to-N 56.3, but had C-to-N ratios around 20 as a consequence of high N content in the organic layer. The German sites receive Norway spruce litter with C-to-N of 38.1 and especially at Schluchsee have lower contents of both C and N in the organic layer. Subsequently the German sites had C-to-N ratios of 27.4 (Schluchsee) and 31.6 (Villingen). Schluchsee has abundant earthworms and a mull/moder organic layer, resulting in the lower total C. As previously noted, catalase activity associated with needle litter was greater at the German sites, despite their lower pH values. Remarkable di€erences were found for tannin contents in the organic layers (Table 4), which were nearly 8-fold greater at the Canadian sites. 3.2.

13

C CPMAS NMR spectroscopy

NMR spectra were interpreted based on previous studies of litter and humus (Wilson et al., 1983; Zech et al., 1987, 1990, 1992; KoÈgel et al., 1988; NordeÂn and Berg, 1990; KoÈgel-Knabner et al., 1992; de Montigny et al., 1993; Preston et al., 1994, 1997; Baldock and Preston, 1995; Trofymow et al., 1995; Wachendorf, 1998; Preston, 1999). The spectra of fresh needle litters are shown in Fig. 2 for black spruce and in Fig. 3 for Norway spruce.

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

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Distinct features for lignin and tannin can also be distinguished in the DD spectra. In this experiment, signals are rapidly lost from carbons with attached hydrogens, except those for which the dipolar interaction is weakened by molecular motion. Therefore, the peaks remaining in the DD spectra represent either groups with quaternary carbons or those with some motion in the solid state, such as methyl, methoxyl, acetate and CH2 in long chains. Condensed tannins give a characteristic broad peak at 105 ppm in the DD spectrum, a region which is otherwise generally free of interferences (Wilson and Hatcher, 1988; Preston et al., 1997). The DD spectra of both litters show this peak, as well as a sharp feature for methoxyl of lignin at 56 ppm. In the phenolic region, tannin features are more distinct than in normal spectra, due to di€erential dephasing of tannin and lignin carbons. Norway spruce has a larger splitting of the peaks at 145.0 and 154.4 ppm consistent with its higher tannin content, with a shoulder at 148 ppm (lignin). For black spruce, the sharp peak at 145.3 ppm has a weak shoulder at 148.3 ppm and a

Fig. 2. Normal and DD-TOSS 13C CPMAS NMR spectra of fresh and decomposed black spruce needle litter.

For the litter spectra, the alkyl intensity (0±50 ppm) comes mainly from surface waxes and cutin and the sharp O- and di-O-alkyl peaks at 72 and 105 ppm from carbohydrates. Tannins and lignins are the main contributors in the aromatic and phenolic regions. Most peaks for condensed tannins are coincident with those from other biopolymers. However, they have a characteristic split peak at 144 and 154 ppm in the phenolic region, compared to guaiacyl lignin which has a peak at 147 ppm with a shoulder at 153 ppm and a methoxyl signal at 55±57 ppm. Both litters have a methoxyl signal at 56 ppm, while the partially split phenolic region is characteristic of a mixture of lignin and tannin. Norway spruce has two broad peaks at 145 and 154 ppm, while for black spruce the phenolic region has a peak at 145.6 ppm with a shoulder at 152.7 ppm. For Norway spruce litter, the more distinct splitting in the phenolic region and the chemical shift of 154 ppm compared to the shoulder at 152.7 ppm for black spruce is consistent with its higher tannin content.

Fig. 3. Normal and DD-TOSS 13C CPMAS NMR spectra of fresh and decomposed Norway spruce needle litter.

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K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

more distinct shoulder at 153 ppm. Di€erences in the phenolic region have been emphasized to highlight features arising from tannin versus lignin structures, though in general, the spectra of both species were very similar. The spectra were also similar to those reported for needle litter by Zech et al. (1987), KoÈgel et al. (1988), NordeÂn and Berg (1990), KoÈgel-Knabner et al. (1992) and Preston et al. (1994, 1997). The relative intensities and alkyl-to-O-alkyl ratios of the 13C CPMAS NMR litter spectra are shown in Table 6. For fresh litter, the main di€erences were the higher alkyl C in black spruce, while Norway spruce was higher in O-alkyl C with a lower alkyl-to-O-alkyl ratio. During decomposition, an increase of intensity in the alkyl region was observed for both species with the greatest changes at Schluchsee. However, a decrease of O-alkyl C commonly associated with decomposition was only seen for Norway spruce litter with strongest changes at the German site Villingen. For black spruce needle litter, the relative intensity of the O-alkyl region remained essentially unchanged. Therefore the increase in the alkyl-to-O-alkyl ratio was mainly due to a relative increase in alkyl C. Sitedependent di€erences in changes of the relative intensities in the other regions of the 13C CPMAS NMR spectra were small and inconsistent, although the relative intensity of the phenolic and carboxyl regions usually decreased. The main features of the spectra (peak position, lineshape) were also generally unchanged by 10 or 12 months of decomposition, as shown by the representative spectra in Figs. 2 and 3. There was an increase in the relative intensity of the 33 ppm peak for black spruce litter at Lake Nipigon. This peak comes from

carbon in long CH2 chains of a more rigid nature, while those at 30 ppm are more mobile (KoÈgel-Knabner et al., 1992). However, it was not associated with a large increase of alkyl C and constitutes a very small proportion of the total alkyl C area. For Norway spruce, the splitting of the phenolic region became less distinct, with loss of the intensity due to tannins at 154 ppm. NMR spectra of the organic layers are shown in Fig. 4 and relative areas are given in Table 7. Spectra from the German sites, especially from Villingen, are similar to the spectra of the decomposed Norway spruce litter (Fig. 3) but with the features much broadened. By contrast, spectra of the organic layers from the two Canadian sites have higher resolution than the fresh litters, especially the peaks for long-chain CH2 at 33 ppm and for tannins at 145 and 155 ppm. All organic layers have peaks at both 30 and 33 ppm, for more mobile and more rigid CH2, but for the Canadian sites, the peak at 33 ppm is much higher than that at 30 ppm. The Canadian samples also have a more intense and well-resolved peak at 130 ppm in the aromatic region. All sites have a partially resolved peak or shoulder at 55±57 ppm. The DD spectra are consistent with tannin structures in all four organic layers (broad peak at 105 ppm). However, the Canadian samples are remarkable in having almost no methoxyl signal in the DD spectrum, whereas it is present in the German samples. For the Canadian sites, the lack of the methoxyl signal, the large splitting of the phenolic region and the relatively sharp peak at 130 ppm in both the normal and DD spectrum indicate a high tannin content (consistent with the chemical assay), and almost no lignin. For

Table 6 Relative intensities (percent of total area) of the 13C CPMAS NMR spectra of black and Norway spruce needle litter: intact litter and after decomposition (10 months in Germany, 12 months in Canada); alkyl/O-alkyl ratios Range (ppm)

Alkyl/O-alkyl

0±50

50±60

60±93

Black spruce Norway spruce

22.5 17.5

5.0 5.4

39.0 42.6

Schluchsee Villingen Black Sturgeon Lake Nipigon

26.9 26.0 26.7 24.3

6.9 6.1 5.1 5.7

39.9 38.9 37.9 37.8

Schluchsee Villingen Black Sturgeon Lake Nipigon

25.1 20.8 19.0 21.2

6.9 5.7 5.4 6.4

39.5 37.7 38.8 40.8

93±112

112±140

Intact litter 10.4 10.8 12.5 10.5 Black spruce decomposed 9.9 8.5 9.6 9.3 10.0 10.0 10.3 10.9 Norway spruce decomposed 9.9 9.0 11.1 11.3 11.3 11.9 11.5 10.7

140±165

165±190

6.9 7.3

5.4 4.2

0.58 0.41

4.5 5.8 5.7 6.0

3.4 4.3 4.6 5.0

0.67 0.67 0.70 0.64

5.3 7.1 7.4 5.7

4.3 6.3 6.2 3.7

0.64 0.55 0.49 0.52

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

787

even though there is little di€erence in relative areas. The German sites are higher in O- and di-O-alkyl C and lower in alkyl C, but the di€erences are small. 4. Discussion 4.1. Comparison of spruce litter chemistry

Fig. 4. Normal and DD-TOSS layers.

13

C CPMAS NMR spectra of organic

the German organic layers, there is a methoxyl peak in the DD spectra and the phenolic region is consistent with a mixture of lignin and tannin. The German samples also have a broad peak at 105 ppm in DD spectrum, indicating tannin, although the chemical assay of the tannin content is much lower (0.35 and 0.5%). Spectra from the German and Canadian sites indicate considerable di€erences in humus quality, Table 7 Relative intensities (percent of total area) of the

Litter species di€er in several properties that have been related to litter quality and readiness to decompose (Tian et al., 1995; Trofymow et al., 1995; Handayanto et al., 1997; Mafongoya et al., 1998; Moore et al., 1999). On the positive side, the Norway spruce litter had a higher N content and lower C-to-N ratio and its NMR spectrum indicated that it was higher in carbohydrates and lower in cutin and surface waxes than was the black spruce. On the other hand, it was higher in total and extractable tannins, and the latter has been linked to protein-binding and inhibition of N mineralization (Handayanto et al., 1997). Several factors need to be considered in interpreting foliar (and humus) tannin analyses. First, foliage tannin contents are a€ected by environmental factors and tend to increase with stresses such as nutrient limitation and insect attack (Ricklefs and Matthews, 1982; Tiarks et al., 1988; Northup et al., 1995a). The sampling site for Norway spruce litter is a typical Mgde®cient site (Raspe et al., 1998). Therefore, the tannin contents of the study litters should not be regarded as standard values for these species. Second, a variety of methods and standards have been used to extract and assay foliar tannins or total (poly)phenolics. We used a highly ecient extractant for condensed tannins (acetone/water) and the PA assay which is speci®c for condensed tannins and was calibrated against a wellcharacterized condensed tannin extracted from balsam®r tips. However, the response in the PA assay is dependent on tannin structure (chain-length and branching) and foliar tannins may comprise a range of sizes and solubilities, from monomers (e.g. catechin and epicatechin, not detected in the PA assay) to structures that are insoluble due to either high molecular weight or cross-linkages to other biopolymers. How-

13

C CPMAS NMR spectra of organic layers

Range (ppm)

Schluchsee Villingen Black Sturgeon Lake Nipigon

0±48

48±90

90±110

110±137

137±160

160±187

187±210

16.9 18.3 19.5 22.5

39.5 42.9 38.8 37.7

12.5 12.4 10.8 10.4

12.6 11.9 13.4 12.6

8.1 6.4 7.7 7.5

7.5 6.7 8.0 7.6

2.9 1.4 1.8 1.7

788

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

ever, preliminary analysis of tannin extracted from black spruce foliage (unpublished) showed similarity in chain length and PA color development to the balsam®r tannin and the NMR spectra were consistent with the higher tannin contents measured in Norway spruce litter. Therefore, in the interpretation of the results, comparisons within species are more reliable than comparison between species and the absolute numbers should be used with caution, especially in relation to other studies. The PA assay of senescent needles yielded 25.3 mg gÿ1 condensed tannins for black spruce needles from the Black Sturgeon site (Canada) and 37.8 mg gÿ1 from Norway spruce needles from Schluchsee (Germany) (Table 4), with the Norway spruce needles having a higher proportion of extractable tannin. These results may be compared to other studies of senescent needles, with the aforementioned cautions. Brown Norway spruce needles from two high-elevation sites in France had 18 and 24 mg gÿ1 total phenolics, based on the Folin±Ciocalteau assay of aqueous extracts (Gallet and Lebreton, 1995). The Folin±Dennis assay of hot water extracts yielded 1.2±4.5% total phenolics for litter from cedar (Thuja plicata Donn.) and hemlock (Tsuga heterophylla (Raf.) Sarg.)/®r (Abies amabilis (Dougl.) Forbes) mixtures from northern Vancouver Island (Keenan et al., 1996). Yavitt and Fahey (1986) found 4% total phenolics in lodgepole pine (Pinus contorta Dougl.) litter using aqueous methanol and the Folin assay. Tiarks et al. (1992) measured 5.7% extractable and 1.7% residual condensed tannins in shortleaf pine (Pinus echinata Mill.) using similar methods to this study and a standard from the same species. Even with the great variety of analytical approaches, tannin contents in the senescent foliage of both species in this study are similar to those reported elsewhere for Norway spruce and other conifers. While the black spruce litter had lower quality based in its total N, C-to-N ratio and NMR spectra, it was lower in tannins and particularly in extractable tannins than the Norway spruce. Therefore, it is unlikely that the elevated tannin contents in the black spruce humus derive from unusually high tannin content in foliar litter. 4.2. Spruce litter decomposition At the end of this short-term decomposition study, there was a loss of 19.4±28.1% of litter mass, 66±79% of total tannins and 76±89% of total tannins. The rapid loss of condensed tannins within 1 year, especially within the ®rst 6 months, is consistent with other reports. Again, direct comparison of results is dicult, because of the wide variety of methods, including assays of total phenolics, as well as more speci®c tests for protein-binding activity and con-

densed and hydrolyzable tannins. However, despite this variety, losses are generally in the range of 80% within one year for litter of both deciduous species (Wilson et al., 1983; Baldwin and Schultz, 1984; Nikolai, 1988; Racon et al., 1988; Scho®eld et al., 1998) and conifers (Wilson et al., 1983; Yavitt and Fahey, 1986; Tiarks et al., 1992; Keenan et al., 1996). Both site and species e€ects were found in this study. After decomposition for 10 months (Germany) and 12 months (Canada), Norway spruce litter always had less mass loss than black spruce litter, and higher concentrations of total and extractable tannins. Concentrations of residual tannin in Norway spruce were also higher at all sites except for Lake Nipigon, where the values for the two species were very close. Since other factors indicated higher litter quality for Norway spruce, it is possible that its higher initial tannin content (especially of extractable tannins) inhibited decomposition. Another possible in¯uence would be di€erences in needle toughness and permeability (Gallardo and Merino, 1993); however, the NMR spectra indicate a lower content of cutin and surface waxes in Norway spruce. In the comparison of sites, the highest tannin losses for both species at both 6 and 10/12 months were found at the German site Villingen. Compared to the Canadian sites, the German sites had higher losses of total and extractable tannins, but losses of residual tannins were similar (tannin losses were still less at the Canadian sites, despite the longer second sampling period). Mass losses within Germany were consistently higher at Villingen than at Schluchsee. At the Canadian sites, mass losses tended to be larger at Lake Nipigon after 6 months, but larger at Black Sturgeon after 12 months. There are large di€erences in the conditions for decomposition among the four sites. According to Cortez (1998), litter decomposition is controlled by earthworm activity as well as by climatic conditions and their e€ects on soil moisture and temperature. Rates of tannin loss were also dependent on site conditions, although these were not speci®ed (Baldwin and Schultz, 1984). Compared to Villingen, Schluchsee has a slightly lower mean annual temperature but higher precipitation. Also, at Schluchsee Lumbricidae are abundant (Lamparski, 1985), whereas at Villingen this is the case only to a minor extent. The mesh size of 1 mm used in this litter bag study allows penetration of small earthworms into the bags. However, the main reason for the slightly lower mass and tannin losses at Schluchsee compared to Villingen was probably the unusually low precipitation at Schluchsee until the ®rst sampling in Spring 1996 (only 63% of the annual wintertime mean; Armbruster, 1998). Probably, the fast initial loss of tannins at Villingen then makes the litter more attractive to decomposers (Harrison, 1971;

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

SÏlapokas and Granhall, 1991; Keenan et al., 1996), which is also indicated by the highest catalase activity of all sites (Fig. 1). The inhibition of catalase by tannins was also observed by Ladd and Butler (1975). Compared to the German sites, the Canadian sites are colder and drier, with no earthworms and lower catalase activity, although the pH is higher. The percentage losses of tannins were lower at the Canadian sites, for both species, tannin fractions and decomposition times (despite the slightly longer ®eld exposure time for the Canadian sites). The more favorable climatic conditions and presence of earthworms at the German sites is probably responsible for the higher tannin losses and catalase activities. Satchell and Lowe (1967) showed that tannin degradation by microorganisms is associated with an increase in the earthworm population in the litter and Tian et al. (1995) found that millipedes and earthworms were important to the breakdown of plant residues of low quality (high C-to-N ratio, lignin and polyphenol contents). In the NMR spectra, decomposition resulted in a relative increase of alkyl versus O-alkyl C, while aromatic, phenolic and carboxyl C content showed only small and inconsistent changes. This pattern is similar to results from other litterbag studies of needle litter (Wilson et al., 1983; Zech et al., 1987; NordeÂn and Berg, 1990). At the end of the decomposition experiment, the highest relative content of alkyl C for both species was at Schluchsee. There were only small changes in the relative intensity of the O-alkyl region (60±93 ppm); black spruce showed essentially no decline, while the greatest decrease was found for Norway spruce at Villingen (42.6 to 37.7%). 4.3. Organic layers and decomposition processes Large di€erences in the properties of the organic layers all suggest development of a pattern of restricted decomposition at the Canadian sites. Humus at both Canadian sites had higher contents of C and N and lower C-to-N ratios than the German sites. The latter would normally be associated with a greater degree of decomposition, but in this case probably indicates low nutrient availability. With increasing stand age, northern black spruce forests tend to show declining nutrient availability at the same time as N is increasingly sequestered in the forest ¯oor. Several factors may contribute to this, including the cold climate, small annual litter inputs of low N content, lack of earthworm activity and an increasing sequestration of N in recalcitrant organic structures and in understorey feathermoss (Pastor et al., 1987; Smith et al., 1998). However, it does not appear to arise from any unusually recalcitrant organic composition of black spruce litter. In this 1-year litterbag study, black spruce lost more mass than Norway spruce at each site and com-

789

parable proportions of tannin. In a multisite Canadawide long-term litterbag study, black spruce litter mass loss after 3 years was 43.9%, the highest of ®ve conifer species (Moore et al., 1999). The 13C CPMAS NMR spectra of the organic layers from the German sites are similar to those in many reports (Zech et al., 1987, 1990, 1992; KoÈgel et al., 1988; KoÈgel-Knabner et al., 1992; de Montigny et al., 1993; Preston et al., 1994; Baldock and Preston, 1995; Wachendorf, 1998; Preston, 1999). Spectra from the Canadian sites have similar peak positions and only slight di€erences in relative areas. However, the much sharper features of the Canadian sites indicate less transformation of the original biopolymers. In particular, the sharp peak at 33 ppm comes from accumulation of long-chain CH2 from cutin, suberin and plant waxes although microbial biomass may also contribute in this region. The NMR spectra also show unusual accumulation of condensed tannins, and a remarkable lack of signals due to lignin. By comparison, spectra of organic layers from the German sites have broader features and a normal mixture of lignin and tannin-derived structures. Little is known about the mechanism of tannin loss from litter and the fate of tannins in humus. There is evidence that both tannins and simpler phenolics are lost by leaching (Kuiters and Sarink, 1986; Yavitt and Fahey, 1986; Gallet and Lebreton, 1995; Scho®eld et al., 1998), which is consistent with the rapid loss of extractable tannins in this study. There are few studies of tannins in humus, for which comparison is again dicult because of the variety of methods used. However, these generally agree in showing low concentrations of 1% or less in humus (Kuiters and Denneman, 1987; Gallet and Lebreton, 1995; Preston, 1999) and Scho®eld et al. (1998) were unable to detect or recover tannins in the mineral soil of a microcosm experiment. In our study, organic layers from Norway spruce sites in Germany had 3.5 and 5.0 mg tannins gÿ1, similar to the low values reported elsewhere. However, the Canadian black spruce sites had much higher amounts (28.7 and 37.6 mg gÿ1) with NMR supporting the chemical assay, despite their receiving litter with lower tannin content. The link between accumulation of tannins and inhibition of N availability may be through formation of tannin±protein complexes (Howard and Howard, 1993; Northup et al., 1995b; Handayanto et al., 1997; Mafongoya et al., 1998). However, such complexes have not been unequivocally detected or isolated in litter or humus, while tannin-rich fractions extracted from humus under salal (Gaultheria shallon Pursh) were all depleted in N (Preston, 1999). Scho®eld et al. (1998) found no evidence for the formation of tannin± protein complexes in willow (Salix exigua Nutt.) litter. The lack of accumulation of tannins in humus and

790

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

mineral soil indicates that under most circumstances their microbial decomposition is not unduly hindered, as would be expected for a major biopolymer component of trees and shrubs (Grant, 1976; Deschamps, 1982; Gamble et al., 1996). It is, therefore, unlikely that the humus tannin accumulation at the Canadian sites can be attributed to either high litter tannin content or an inherent resistance of black spruce litter to decomposition. It was not possible in this study to determine whether the tannin and N accumulations were linked. A high proportion of the humus tannin was extractable and, therefore, not likely to be sequestered in insoluble tannin±protein complexes. It may be that tannins, like alkyl C, can accumulate under certain conditions where decomposition is hindered, as was found to a lesser extent for humus from sites of contrasting productivity on northern Vancouver Island (de Montigny et al., 1993). As striking as the accumulation of tannins, is the unusual depletion of lignin structures in humus at the Canadian sites. Further studies are necessary to ascertain the spatial and temporal extent of these phenomena in black spruce ecosystems, and whether even temporary accumulations of humus tannin may contribute to long-term reduction of N availability. 5. Conclusions Condensed tannins in black and Norway spruce needle litter decreased quickly during decomposition for up to 1 year at the Canadian and German sites. Greater mass losses were observed for black spruce needles despite less favourable C-to-N ratios and higher contents of surface lipids and cutin than Norway spruce needles. The higher initial tannin contents in Norway spruce needle litter, especially of the extractable fraction, may have been responsible for the slower decomposition. The results also indicate a strong in¯uence of climate on early stages of decomposition. Humus at both Canadian sites had extremely high tannin contents and was poorly decomposed, despite having lower C-to-N ratios and higher N contents that would normally indicate more advanced decomposition. Coincident with the accumulation of organic N (typical of nutrient-limited black spruce ecosystems), condensed tannins and long-chain CH2 structures, was a unique depletion of structures characteristic of lignin. Di€erences in needle litter quality, cannot account for such di€erences in the humus. Further investigations are needed to understand the di€erences of decomposition pathways in the Canadian and German sites. These may arise from di€erences in other inputs (bark, wood, roots, understorey veg-

etation) and in site properties (climate, decomposer community, earthworm activity). As the Canadian sites accumulate both tannins and poorly available N, it is important to understand whether tannins per se are a critical factor in the development of restricted decomposition and nutrient limitation in black spruce ecosytems.

Acknowledgements We gratefully acknowledge the reliable technical assistance of Ulrike Benitz, Kevin McCullough, Gary Koteles, Pamela Perreault and Daniela Wohlfahrt. We thank the BMBF (German Ministry of Science and Technology, Bonn) for funding and the GKSS and DLR for coordinating visits of German scientists in Canada. Canadian travel to Germany was funded through The Going Global Program for Science and Technology of the Department of Foreign A€airs and International Trade, Canada.

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