Effects of tree species and clear-cut forestry on forest-floor characteristics in adjacent temperate forests in northern Spain

Effects of tree species and clear-cut forestry onforest-floor characteristics in adjacent temperateforests in northern Spain

Nahia Gartzia-Bengoetxea, Ander Gonzalez-Arias, and Inazio Martınez de Arano

Abstract: The litter layer (L) and fermented humified layer (FH) in forest floor under European beech (Fagus sylvaticaL.), pedunculate oak (Quercus robur L.), and radiata pine (Pinus radiata D. Don) were studied in northern Spain. Recov-ery from heavy mechanization was also assessed in a chronosequence from two adjacent pine plantations (3 and 16 yearsold). Interspecific differences in forest-floor mass and mass distribution were related to litter quality and decompositionrates. In the L layer, acid-insoluble residue/N, C/N, and C/P ratios were lower, and pH and Ca contents higher in the de-ciduous stands than in the pine stand. The microbial respiration rate was higher and functional diversity lower in the pinestand than in the deciduous stands, without differences in microbial biomass. Cross-polarization, magic angle spinning 13Cnuclear magnetic resonance and proximate analysis revealed similar C functional groups in beech and pine stands. Forestfloor was still absent 3 years after heavy mechanization, and after 16 years, it was 50% less abundant than in the maturestand. Microbial respiration rate, biomass, and diversity were similar in the L layer in 16-year-old and mature pine stands,but in the FH layer, microbial-community diversity remained low after 16 years. The results underline the effects of forestmanagement on C transformations in the forest-floor layers. These effects may be evident even after 16 years of heavymechanization.

Resume : Les horizons de litiere (L) et de litiere fermentee et humifiee (LH) de la couverture morte presente sous le hetreeuropeen (Fagus sylvatica L.), le chene pedoncule (Quercus robur L.) et le pin de Monterey (Pinus radiata D. Don) ontete etudies dans le nord de l’Espagne. La recuperation a la suite d’une mecanisation intensive a egalement ete evalueedans une chronosequence constituee de deux plantations adjacentes de pin agees de trois et 16 ans. Des differences inter-specifiques dans la masse et la distribution de la masse de la couverture morte etaient reliees a la qualite de la litiere et autaux de decomposition. Dans l’horizon L, les rapports residus solubles dans l’acide/N, C/N et C/P etaient plus faibles et lepH ainsi que le contenu en Ca etaient plus eleves dans les peuplements feuillus que dans le peuplement de pin. La respira-tion microbienne etait plus elevee et la diversite fonctionnelle etait plus faible dans le peuplement de pin que dans lespeuplements feuillus sans qu’il y ait de differences dans la biomasse microbienne. La technique CPMAS 13C RMN et l’an-alyse immediate ont revele que les groupements fonctionnels de C etaient semblables dans les peuplements de hetre et depin. La couverture morte etait toujours absente trois ans apres une forte mecanisation et, apres 16 ans, elle etait 50 %moins abondante que dans le peuplement mature. Le taux de respiration, la biomasse et la densite microbiennes etaientsemblables dans l’horizon L des peuplements ages de 16 ans et des peuplements matures de pin mais, dans l’horizon LH,la diversite de la communaute microbienne restait faible apres 16 ans. Les resultats mettent en evidence les effets del’amenagement sur les transformations de C dans les horizons de la couverture morte. Ces effets peuvent etre evidentsmeme 16 annees apres une forte mecanisation.

[Traduit par la Redaction]

IntroductionDuring the twentieth century, large-scale plantations of

the exotic Pinus radiata D. Don were established on aban-doned farmland and mountainous land in the temperate Bas-

que Country (northern Spain) (Michel 2003). The speciesnow dominates forest plantations in the region and forestsare currently being managed in a clear-cut regime with rota-tion lengths between 30 and 40 years, harvesting with chain-saw and skidding, and mechanical site preparation prior toplanting, by processes such as blading and downslope rip-ping. These human-induced changes to the landscape arelikely to cause changes in forest composition, net primaryproduction, and consequently, regional patterns of carbon(C) cycling (Pastor and Post 1988; Finzi et al. 1998; Yanaiet al. 2000). Litter decomposition and the subsequent releaseof nutrients in plant-available forms are essential processesin the functioning of forest ecosystems. The rate of litter de-composition is determined by the prevailing environmentalconditions in a given location (Prescott 1995), by the intrin-sic chemical properties of plant litter or litter quality (Al-mendros et al. 2000), and by the diversity and activity of

Received 6 October 2008. Accepted 3 April 2009. Published onthe NRC Research Press Web site at cjfr.nrc.ca on 7 July 2009.

N. Gartzia-Bengoetxea,1 A. Gonzalez-Arias, andI. Martınez de Arano.2 Forestry Unit, NEIKER-Tecnalia(Basque Institute for Agricultural Research and Development),Zamudio Technology Park 812, E-48160 Derio, Bizkaia, BasqueCountry, Spain.

1Corresponding author (e-mail: [email protected]).2Present address: Southern Europe Forest Owners Union(USSE), Larrauri 1-B, E-48160 Derio, Bizkaia, BasqueCountry, Spain.

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Can. J. For. Res. 39: 1302–1312 (2009) doi:10.1139/X09-053 Published by NRC Research Press

the microbial community (Leckie et al. 2004). Under similarenvironmental conditions, substrate quality and the nature ofthe decomposer community are directly related to the bio-logical diversity (Hattenschwiler et al. 2005).

Different tree species show different patterns of forest-floor accumulation, owing to differences in soil organic mat-terchemistry (Raulund-Rasmussen and Vejre 1995; Hannamet al. 2004). In a study of Mediterranean evergreen shrubs,Quideau et al. (2005) found that the leaf litter under scruboak was dominated by carbonyl C, whereas under manzanita(Arctostaphylos manzanita Parry) it was dominated by O-alkyl C and under coniferous vegetation by alkyl C. Toour knowledge, differences in the chemical nature and dis-tribution of the organic constituents of litter, such as carbo-hydrates, phenols, and carboxylic acids, have not beeninvestigated in temperate forests. These components affectthe chemical stability of accumulated organic matter andexert a strong influence on soil microbial activity by func-tioning as sources of energy for the growth and activity ofdecomposing organisms (Goh and Heng 1987).

Despite differences in patterns of accumulation under dif-ferent tree species, inputs are balanced by outputs in the for-est floor of mature forests. However, this equilibrium isdisrupted by clear-cut forest harvesting and subsequent soilpreparation (Covington 1981). Such practices alter the quan-tity and biochemical composition of plant litter, whichserves as the primary substrate for microbial activity. Theamount of dead organic matter on the soil surface afterharvesting and site preparation depends on the forest-management practices selected. Stem-only harvesting mayleave an abundance of dead organic matter, although inputsof detritus decrease thereafter. However, during forest prac-tices, such as blading and subsequent down-slope ripping,all aboveground organic residues are pushed out of thestand with the front blade of a bulldozer (Olarieta et al.1997; Merino et al. 2004). Furthermore, postharvest or-ganic remains are biochemically different from the fineroots, leaves, and dead plant tissues that comprise litter inmature forests (Covington 1981). Such differences in sub-strate input may alter the composition of soil microbialcommunities because bacteria, actinobacteria, and fungivary in their physiological abilities to metabolize organicsubstrates contained in plant litter (Paul and Clark 1996).Borman and Likens (1979) suggested that the rate of nu-trient mobilization from the forest floor may regulate re-covery after devegetation, by ensuring the availability ofessential elements during recovery. The forest floor playsan important role in recovery after disturbance (Covington1981) and the effect of forest harvesting on soil organicmatter is important in terms of not only local successionalprocesses but also global C storage (Yanai et al. 2000).

In this context the present study was designed to providebasic information on the forest floor in temperate semina-tural and cultivated forests. The main objectives were (i) tocompare biotic and abiotic characteristics of the forest floorin three stands dominated by different tree species (Euro-pean beech, pedunculate oak, and radiata pine) developedunder similar conditions of soil parent material, geomorphol-ogy, and climate, (ii) to determine the effects of clear-cutting and mechanical site preparation on the forest floorin a 3-and 16-year-old radiata pine stand, and (iii) to ex-

plore the relationships among the characteristics of theseforest floors to gain insights into the effect of specieschange and intensive mechanization on C dynamics.

Materials and methods

Forest-floor samplingThe study site (30T 534075 4783284) was selected as an

example of the temperate forest landscape in the BasqueCountry (northern Spain); the types of soil (Typic Udor-thent; Soil Survey Staff 2006) and climatic conditions(mean annual temperature 14.1 8C and precipitation1200 mm) were similar in the stands under study. The aimof the study was to evaluate three representative stands oftwo mature seminatural forests, English oak (Quercus roburL.) and European beech (Fagus sylvatica L.) (hereinafter re-ferred to as oak and beech) and a nearby first-rotation andnonmechanically cultivated radiata pine (Pinus radiata D.Don) plantation (hereinafter referred to as 40 year pine) toevaluate the effect of change in tree species on the forestfloor in these stands. Density in the mature stands was ap-proximately 300 trees�ha–1, although basal area was largerin the mature pine stand (90 m2�ha–1) than in the oak andbeech stands (64 and 63 m2�ha–1, respectively). In addition,a chronosequence was sampled from two adjacent clear-cutsites (3 and 16 years old, hereinafter referred to as 3 and16 year pine) to evaluate the effect of mechanized forest op-erations 3 and 16 years after disturbance. The density of the16 year pine stand was 1010 trees�ha–1 and the basal area18 m2�ha–1. The 3 year pine stand was established using a3 m � 2 m grid, and at the time of sampling the meanheight was 1.2 m. Forest operations in the two younger pineplantations included harvesting with skidders and site prepa-ration by blading and downslope ripping. In blading, the res-idues of the previous plantation and competing vegetationshould be removed without disturbing the mineral soil sur-face. However, sometimes the upper centimetres of soil arealso excavated and slash and surface organic material aredisplaced to form small piles downslope. Downslope rippingconsists of deep ploughing (&50 cm) following the maxi-mum slope of the stand. All the studied stands faced southand were developed on slopes with similar steepness.

In each stand (stands varied in area from 0.8 to 1.5 ha),three trees were selected at random and forest-floor samples(10 samples, 15 cm � 15 cm template) were systematicallycollected within a 2.5 m radius from the base of each ofthese trees, on 13 April 2005. All samples were collected atthe same time and divided into L and FH layers and com-bined to give one composite sample per tree and layer. TheL layer consisted of slightly decomposed litter of identifi-able origin, and the FH layer, which we were not able toseparate into F and H layers, consisted of partially andhighly decomposed organic material. Samples for chemicalanalysis were oven-dried to constant mass, weighed, andground (<0.5 mm) in a laboratory mill, and samples for mi-crobiological analysis were stored at 4 8C for no more than7 days. The L-layer samples were cut into smaller pieces(&1 cm2) and the FH-layer samples were passed through a5 mm mesh sieve.

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Chemical, biochemical, and moisture analysis of forestfloor

Organic C and total nitrogen (N) were analyzed in aLECO CNS 2000 (LECO Corporation, Michigan, USA). To-tal concentrations of phosphorus (P), calcium (Ca), magne-sium (Mg), potassium (K), and sodium (Na) weredetermined in aqueous extracts after wet digestion with ni-tric acid – perchloric acid by inductively coupled plasmaemission spectroscopy (Varian Iberica S.L., Barcelona,Spain). Forest-floor pH was determined in 1:10 forest floor :water solution (Blakemore et al. 1977). The water-solublecarbohydrates were determined colorimetrically by theanthrone method in water extracts, according to Deriaz(1961). Acid-detergent fibre (ADF) and acid-insoluble resi-due (ADR) were determined with a ANKOM220 fiber ana-lyzer (Ankom Technology Corporation, Macedon, NewYork, USA) (Van Soest et al. 1991). The cellulose fraction(CEL) was estimated as ADF – ADR. Ash was determinedafter 4 h at 550 8C. The moisture content of each samplewas determined after drying to constant mass at 105 8C.

Solid-state cross-polarization, magic angle spinning 13Cnuclear magnetic resonance (CPMAS 13C NMR)spectroscopy

The solid-state 13C NMR spectra of the L and FH layersof oak, beech, and 40 year pine were obtained with a BrukerDSX 300 spectrometer (Bruker Instruments, Karlsruhe, Ger-many) operated at a 13C resonance frequency of 75.49 MHz.All 13C NMR spectra were acquired by cross-polarization.Dry, powdered samples were spun at 4.7 kHz in a 7 mmoutside diameter rotor. The 1H 908 pulse length was 4.5 ms,the recycling time was 2 s, and the contact time was 1 ms.The number of scans was approximately 30 000 and the re-sults were processed by 30 Hz line-broadening and baselinecorrection. Chemical shifts are reported relative to tetrame-thylsilane at 0 ppm, as reported by Lorenz et al. (2000),and were integrated to determine the percentage of totalarea (relative intensity).

Activity and quantity of the microbial community inforest floor

C mineralization of forest floors was determined in thelaboratory. Three replicate subsamples of each (fresh) sam-ple (5 g) were incubated at 28 8C for 28 days in airtightglass jars (0.5 L). The CO2 emitted was trapped in 10 mLof 1 mol�L–1 NaOH containers and determined on days 1, 2,3, 8, 14, 22, and 28 by titration with HCl to the phenol-phthalein endpoint, after precipitation of carbonates withBaCl2. The rate of C mineralization or basal respiration wasexpressed as microbial respiration rate at the end of the in-cubation period (mg C-CO2�(g forest floor)–1�h–1) and miner-alizable C as cumulative microbial respiration during28 days (mg C-CO2�(g forest floor)–1). Microbial biomass Cwas determined by the fumigation–extraction method, whichwas essentially that described by Vance et al. (1987).Briefly, 2 g of fresh forest-floor material was fumigated for24 h at 28 8C with ethanol-free chloroform vapour. Fumi-gated and unfumigated samples were extracted in 1 : 4 for-est floor : 0.5 mol�L–1 K2SO4 (m/v) (30 min, 200 r�min–1).The extracts were filtered through Whatman 42 filters and

total organic C was determined after digestion of the sam-ples in chromic acid.

Diversity of forest-floor microbial communitiesThe functional diversity of microbial communities was

characterized by means of community-level physiologicalprofiles (CLPP), with a Ecoplate microplating system (Bi-olog, Hayward, California, USA) based on the methods ofDegens et al. (2001) and Grayston and Prescott (2005). Eco-plates contain 31 C substrates that are ecologically relevantcompounds and are likely to be useful for microbial analysisand for detecting microorganisms that are often missed be-cause they are swamped by faster growing r-strategist spe-cies on GN plates (Campbell et al. 1997). Briefly, moistforest-floor samples (5 g) were suspended in 50 mL of one-quarter strength Ringers solution with thirty 3 mm diameterglass beads, and processed in a reciprocating shaker at highspeed (250 r�min–1) for 15 min. Serial 10-fold dilutions ofthe samples (to 10–3) were prepared in deionized water andcentrifuged at 750g for 10 min. The Ecoplates were incu-bated with aliquots (125 mL) of the resulting suspensions,then incubated in the dark at 28 8C, and the colour wasread as light absorbance at 595 nm after 0, 16, 20, 24, 40,44, 48, 64, 68, 72, 88, 92, 96, and 160 h in a multifunctionalZenyth 3100 microplate reader (Anthos Labtec Instruments,Salzburg, Austria). The absorbance value of the control wassubtracted from the absorbance value of wells containing Csubstrates for each time. The average number of substratesused (NUS) was calculated on the basis of the majority-rulesdecision established by Goodfriend (1998) whereby sub-strate use was confirmed if the dye reaction was positive(>0.25 absorbance units); Shannon’s diversity index wasused as a practical indicator of overall microbial functionaldiversity (Degens et al. 2001). Average well colour develop-ment was determined for each sample and used to transformindividual values of C sources to eliminate variation in aver-age well colour development caused by differences in celldensity (Garland and Mills 1991).

Statistical analysisOne-way analysis of variance (ANOVA) was applied to

forest-floor chemical properties to detect differences in theL layers in stands dominated by different tree species. Vari-ables were tested for normality, and Levene’s test for homo-geneity of variances was used prior to conducting one-wayANOVA; a protected least-significant difference (PLSD)test was used to determine the significance of the main ef-fects revealed by ANOVA. In a similar way, Student’s ttest was used to test for differences in the chronosequenceand between forest-floor layers. Replication of forest standsincluded in chronosequences is often difficult because com-parable stands are not always available (Zak et al. 1990;Idol et al. 2002; Howard et al. 2004; Teklay and Chang2008); the same happened with mature stands, thereforepseudoreplicates were considered and statistical analysesperformed to gain a better understanding of the data struc-ture. Differences were considered significant at P < 0.05.

The CLPP data were analyzed first by principal compo-nent analysis (PCA) using a correlation matrix, and compo-nent scores were compared by mean separation tests todetect significant differences between tree species (PLSD)

1304 Can. J. For. Res. Vol. 39, 2009


and the chronosequence studied (t test) and forest-floorlayers (t test).

Another PCA was performed that considered the wholestandardized data set to be an explanatory test of the multi-variate significance of component factors between groups(Stevenson et al. 2004). Factors in the PCA analysis wereretained to explain at least 50% of the variance in the origi-nal data and composite variables of the factors were selectedon the basis of the variable loadings being greater or equalto 0.6 (Lee 1993).

Results

Mass and chemical and biochemical properties of forestfloor

Mature standsTotal forest-floor mass varied with tree species (F[2] =

18.98, P < 0.01). Forest-floor mass in the beech stand(25.2 ± 2.1 Mg�ha–1) was significantly higher than in the40 year pine stand (16.35 ± 1.9 Mg�ha–1) and was lowest inthe oak stand (7.55 ± 0.2 Mg�ha–1). The distribution offorest-floor mass between the layers also varied dependingon tree species. The L layer comprised 100% of forest-floormass under oak, whereas each layer accounted for 50% offorest-floor mass beneath beech.

In the L layers, pH and Ca concentration were signifi-cantly higher in the oak and beech stands than in the pinestand, whereas K and Mg concentrations of were similar inthe beech and 40 year pine stands and significantly lowerthan in the oak stand (Table 1). The total C concentrationwas lower in the oak stand than in the beech and 40 yearpine stands; however, N and P concentrations were similarin the oak and 40 year pine stands and significantly higherin the beech stand. With regard to litter-quality variables,C/N and C/P ratios were similar in oak and beech forests,whereas the same ratios were significantly higher in the ma-ture pine forest. The forest-floor layers differed significantlyin terms of concentrations of nutrients and quality of organic

matter. A significant depth-related reduction in C/N and C/Pratios was observed in the beech and 40 year pine stands,together with a significant depth-related increase in K, Mg,and P concentrations in the 40 year pine stand and a signifi-cant depth-related increase in K and Na concentrations inthe beech stand (Table 1). In the FH layer, the only signifi-cant differences between beech and mature pine stands werethe higher pH and lower C/N ratio in the beech stand.

The biochemical properties of slightly decomposed littermaterial (L layer) were similar in the beech and pine standsbut significantly different in the oak stand. ADF and ADRvalues were significantly lower in the oak stand than in thebeech and 40 year pine stands (Table 2). However, theADR/N ratio, another litter-quality variable, varied signifi-cantly among the oak, beech, and 40 year pine stands, withthe highest value in the pine stand. Similar properties wereobserved in the FH layers in the beech and 40 year pinestands.

In all stands, ADF, CEL, and ADR were significantlylower in the FH layer than in the L layer. Water-soluble car-bohydrates also decreased with depth in all forest floors(Table 2).

Radiata pine chronosequenceForest-floor mass varied significantly with time since dis-

turbance (F[2] = 33.68, P < 0.001). Forest floor was totallyabsent from the 3 year pine stand, mainly as a result of me-chanical site preparation (blading and downslope ripping).Forest-floor mass was greater in the 16 year pine stand(8.13 ± 0.6 Mg�ha–1; mean ± SD) but still significantlylower than in the mature pine stand (16.35 ± 1.9 Mg�ha–1).The distribution of forest-floor mass also differed betweenlayers in the two coniferous stands with forest floor (16and 40 year pine); the FH layer accounted for 70% offorest-floor mass in the 40 year pine and 30% in the 16year pine.

Chemical properties differed between the 16 and 40 yearpine only in the L layer. Both stands showed similar concen-

Table 1. Moisture content and chemical properties of each forest-floor layer (L and FH) under mature stands and the radiata pine chrono-sequence.

L layer FH layer

Oak Beech 40 year pine 16 year pine Beech 40 year pine 16 year pineMoisture content (%) 49.7 (0.01)a 60.1 (0.01)b 56.3 (0.01)b 57.4 (0.01) 65.2 (0.00)a 58.6 (0.01)b 57.3 (0.02)pH 4.67 (0.10)a 5.03 (0.05)a 4.15 (0.10)b 4.06 (0.04) 4.67 (0.12)a 4.04 (0.08)b 4.33 (0.06)C (mg�g–1) 349 (34)a 483 (3)ba 472 (23)ba 493 (3)a 339 (32)b 327 (30)b 317 (27)bN (mg�g–1) 11.8 (0.9)a 18.4 (0.3)b 12.2 (0.5)a 12.2 (0.5) 17.1 (1.7) 12.2 (0.6) 11.5 (0.8)P (mg�g–1) 0.38 (0.00)a 0.52 (0.02)b 0.38 (0.01)aAa 0.55 (0.02)B 0.66 (0.06) 0.45 (0.02)b 0.59 (0.06)S (mg�g–1) 2.22 (0.18)a 3.47 (0.04)b 2.93 (0.06)c 2.95 (0.09) 3.60 (0.48) 3.07 (0.20) 2.28 (0.00)Ca (mg�g–1) 12.59 (1.74)a 9.01 (0.28)aa 4.93 (0.46)ba 4.22 (0.60) 4.68 (0.58)b 2.84 (0.14)b 2.41 (0.24)Mg (mg�g–1) 1.87 (0.24)a 1.17 (0.03)b 0.88 (0.03)ba 0.89 (0.03)a 1.54 (0.11) 1.86 (0.27)b 1.49 (0.10)bK (mg�g–1) 3.75 (0.67)a 1.29 (0.01)ba 0.94 (0.06)ba 1.42 (0.13)a 3.87 (0.47)b 5.08 (0.85)b 3.88 (0.35)bNa (mg�g–1) 0.32 (0.03)a 0.21 (0.01)ba 0.24 (0.00)bA 0.20 (0.00)Ba 0.34 (0.01)b 0.42 (0.05) 0.30 (0.02)bC/N ratio 29.3 (0.8)a 26.2 (0.5)aa 38.9 (2.4)ba 40.5 (1.8)a 19.9 (0.4)ab 26.7 (1.1)bb 27.6 (0.3)bC/P ratio 915 (78)a 941 (47)aa 1257 (74)bAa 894 (34)Ba 517 (14)b 730 (77)b 552 (50)b

Note: The FH layer was absent in the oak stand. Chemical concentrations are expressed on a dry-mass basis. Values are means, with standard deviations inparentheses. Values followed by a different lowercase letter are significantly different (P < 0.05) between mature stands dominated by different tree speciesaccording to the protected least-significant difference test for the L layer and t test for the FH layer. Values followed by a different uppercase letter aresignificantly different (P < 0.05) between stands in the radiata pine chronosequence, according to the t test. Values followed by a different Greek letter aresignificantly different (P < 0.05) between forest-floor layers (L and FH) from each stand, according to the t test.



trations of nutrients and C/N ratios, but the C/P ratio in theL layer in the 16 year pine was significantly lower, owing tothe higher P concentration (Table 1).

Biochemically, in the two pine stands (16 and 40 years)the L layers were very similar, and the only significant dif-ference was that the water-soluble carbohydrate content inthe FH layer was higher in the 16 year than in the 40 yearpine stand (Table 2).

Solid-state CPMAS 13C NMR spectroscopy

Mature standsThe 13CPMAS NMR spectra of the L and FH layers from

mature oak, beech, and radiata pine forests are shown inFigure 1. In the L layers, the ALK region of the spectrashowed a single mean peak at 29–30 ppm, corresponding topolymethylene-type carbons, but oak and beech samplesshowed an additional shoulder at 19–20 ppm, correspondingto acetate groups. For all tree species, major signals in theO-ALK region of carbons were found at around 73 ppm,which is characteristic of the carbons of cellulose and hemi-cellulose. The shoulder at 63 ppm was assigned to the C-6carbon of carbohydrates and was noteworthy in the L layersof the beech and oak forest floor. An additional peak at 56–57 ppm was a C signal for lignins (Fig. 1).

In the DI-O-ALK region of the spectra, the C-2 and C-6carbons of syringyl lignin units probably contributed to thepeak centred around 105 ppm (Preston and Trofymow2000), which was present in all samples. However, the peakat 117 ppm, which may correspond to C-substituted aro-matic carbons (Quideau et al. 2005), was less ambiguous inbeech forest floor, while phenolic carbons between 140 and164 ppm were more intense in the oak forest floor samples.Finally, the peak at 174 ppm, which was indicative of car-bonyl carbons, was also more noteworthy in oak forest floorsamples.

In the spectra of the FH layers, beech and mature pineforests differed mainly in the absence of the peak between116 and 118 ppm in the mature pine forest floor, which wasindicative of aromatic C carbons (AROM). In contrast, thepeak of phenolic carbons, between 140 and 164 ppm, wasmore intense in the pine than in the beech stands. The majordifferences found between forest-floor layers were in the

Table 2. Biochemical properties of each forest-floor layer (L and FH) under different tree species and management practices.

L layer FH layer

Oak Beech 40 year pine 16 year pine Beech 40 year pine 16 year pineASH 10.83 (1.64) 5.68 (0.42)a 5.91 (1.29)a 5.01 (0.34)a 21.58 (2.53)b 16.46 (0.84)b 23.63 (2.91)bADF 44.0 (4.2)a 62.2 (0.7)ba 57.3 (3.5)aba 57.3 (0.8)a 34.2 (3.7)b 34.2 (5.1)b 31.7 (2.1)bCEL 16.0 (1.3) 21.5 (0.7)a 20.2 (1.4)a 20.8 (0.6)a 10.8 (0.9)b 11.7 (1.9)b 11.3 (0.9)bADR 28.0 (3.0)a 40.7 (0.9)ba 37.2 (2.1)aba 36.5 (0.2)a 23.7 (3.1)b 22.6 (3.2)b 20.5 (1.1)bWSC 3.12 (0.29) 3.55 (0.29) 5.78 (1.39) 5.71 (0.24) 2.84 (0.11) 3.06 (0.05)A 6.65 (0.18)BADR/CEL ratio 1.74 (0.1) 1.90 (0.09) 1.85 (0.03) 1.76 (0.04) 2.21 (0.23) 1.95 (0.04) 1.83 (0.05)ADR/N ratio 23.5 (0.8)a 22.1 (0.3)aa 30.7 (2.4)ba 30.0 (1.4)a 13.8 (0.9)b 18.3 (1.6)b 18.0 (0.6)b

Note: The FH layer was absent in the oak stand. Values are percentages (on ash-free dry-mass basis) for ash (ASH), acid-detergent fibre (ADF),cellulose (CEL), and acid-insoluble residue (ADR) and in milligrams per gram (on dry-mass basis) for water-soluble carbohydrates (WSC). Values aremeans, with standard deviations in parentheses. Values followed by a different lowercase letter are significantly different (P < 0.05) among maturestands dominated by different tree species, according to the protected least-significant difference test for the L layer and a t test for the FH layer. Valuesfollowed by a different uppercase letter are significantly different (P < 0.05) between stands in the radiata pine chronosequence, according to the t test.Values followed by a different Greek letter are significantly different (P < 0.05) between forest-floor layers (L and FH) from each stand, according tothe t test.

Fig. 1. CPMAS 13C NMR spectra of the L layers (a) and FH layers(b) from the forest floor in three mature stands (oak, beech, and ra-diata pine).

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mature pine forest, with increasing PHE and CARB contentsin the FH layer. No differences were found between layersin the beech stand (Table 3).

Microbial biomass and C mineralization in the forestfloor

Mature standsMicrobial biomass C (MBC) varied among tree species

only in the FH layer (Fig. 2) and was significantly higher inthe beech stand than in the 40 year pine stand. However,MBC was two times higher in the L layer than in the FHlayer in the beech stand, and three times higher in the40 year pine stand.

In contrast to microbial biomass, microbial respirationrates in the forest floor, an indicator of microbial activity,varied substantially in the L layers among tree species. Therespiration rate in the 40 year pine stand was higher than inthe beech and oak stands. The oak and beech stands released60.2 ± 2.3 and 71.5 ± 10.6 mg C-CO2�(g forest floor)–1�h–1,respectively, while the 40 year pine stand respired 98.8 ±2.5 mg C-CO2�(g forest floor)–1�h–1. No significant differen-ces in microbial respiration rates were found in the FHlayers (Fig. 2). In all stands, microbial respiration rateswere significantly lower in the FH layer than in the L layer(Fig. 2).

The metabolic quotient (qCO2), i.e., the ratio between C-CO2 respiration and MBC did not vary in either the L or theFH layers among mature stands (Fig. 2).

In the L layer, cumulative C was lowest beneath the oakstand and slightly higher and similar in the beech and 40year pine stands (Fig. 3a). Similar respiration patterns wereobserved in the FH layer from the mature pine and beechstands (Fig. 3b). As with microbial biomass and basal respi-ration rate, cumulative C decreased consistently with forest-floor depth.

Radiata pine chronosequenceIn the L layer of both pine stands, MBC, basal respiration

rates, and metabolic quotients of the microbial communitywere very similar (Fig. 2). However, in the FH layers,MBC was significantly lower, and the qCO2 value signifi-cantly higher, in the 16 year pine stand than in the maturepine stand. Cumulative respiration in both layers studiedwas significantly higher in the 16 year pine stand than inthe mature pine stand.

The effect of forest-floor layer was also consistent in the

16 year pine stand. MBC was six times higher in the L layerthan in the FH layer in the 16 year pine stand; the basal res-piration rate and cumulative C were almost two times higherin the L layer and the qCO2 value three times higher in theFH layer than in the L layer.

Diversity of the forest-floor microbial community

Mature standsThe NUS values for the forest-floor L layer varied signif-

icantly depending on the species; of 31 substrates in theEcoplates, oak and beech stands used, on average, 17 and18 substrates, respectively, in 72 h, while the 40 year pinestand used, on average, 11 substrates. The mean NUS valuesin the FH layer were 15 in the beech stand and 11 in the40 year pine stand. In terms of forest-layer effects, microbialcommunities from the L and FH layers did not differ signifi-cantly in NUS value (data not shown).

Shannon’s diversity index for the L layers was signifi-cantly higher in the oak and beech stands than in the maturepine stand (Fig. 4). Microbial community diversity indexvalues In the FH layer were similar in the two maturestands.

The PCA performed with (CLPP explained almost half ofthe total variance with the first three factors. One-way AN-OVAs and t tests of PCA1, PCA2, and PCA3 scores ob-tained from forest floors from mature stands and the radiatapine chronosequence indicated that CLPP did not varyamong mature stands, or within the radiata pine chronose-quence studied. However, the PCA1 and PCA2 scores dif-fered significantly (P < 0.05) between the L and FH layersfrom the beech stand. C sources with high correlation coef-ficients for PCA1 and PCA2 are listed in Table 4.

ANOVA of the utilization of six different groups of Csources (amides, amino acids, carbohydrates, carboxylicacids, phenolic acids, and polymerics) in the CLPP showedthat microbial communities from the L layer of the 40 yearpine stand used fewer amino acids than the oak and beechstands (P < 0.05). Furthermore, the beech stand used signifi-cantly more amino acids in the L layer than in the FH layer(P < 0.05).

Radiata pine chronosequenceThe NUS value for the L layer did not differ between the

40 and 16 year pine stands in either forest floor layers. Of31 substrates in the Ecoplates, in the L layer, the 40 yearpine stand used, on average, 11 substrates, while the

Table 3. Integration of CPMAS 13C NMR spectra of samples from the L andFH layers of forest floor under different tree species.

ALK O-ALK DI-O-ALK AROM PHE CARBOak

L 25.3 48.6 9.8 9.1 3.3 3.9FH

BeechL 20.5 50.7 11.6 10.0 3.8 3.4FH 21.9 47.5 11.3 10.8 4.4 4.1

40 year pineL 20.0 51.4 12.7 9.0 4.1 2.8FH 20.1 46.6 11.3 11.2 6.2 4.7



16 year pine stand used, on average, 12 substrates in 72 h.In the FH layer, the mean NUS values were 11 and 10, re-spectively.

Shannon’s diversity index values differed between the 40and 16 year pine stands only in the FH layer, with the

16 year pine stand showing significantly lower diversity.Furthermore, Shannon’s diversity index values between thetwo forest-floor layers differed in the 16 year pine stand,for which the microbial diversity index value was signifi-cantly lower in the lower forest-floor layer.

The (PCA performed with CLPP data indicated that ca-tabolic profiles of forest-floor communities from matureand clear-cut 16 year pine stands were very similar to theutilization of six different groups of C sources in bothforest-floor layers. There were no differences in the utiliza-tion of these C sources between the L and FH layers in themature pine stand; however, the 16 year pine stand used sig-nificantly fewer amides in the L layer than in the FH layer(P < 0.05) and significantly more carboxylic acids in the Llayer than in the FH layer (P < 0.05).

Relationship between carbon mineralization rates, litterquality, and microbial community

PCA of all forest-floor properties is shown in Fig. 5a. Thefirst three factors explained 75% of the total variance.PCA1, which accounted for almost half of the total variancein the PCA, indicated that the chemical properties of the for-est floor, such as Mg, K, and Na concentrations, were nega-tively correlated with biochemical properties such as ADF,CEL, and ADR. Furthermore, microbial biomass and respi-ration rates were related to litter-quality parameters (C/N,C/P, and ADR/N ratios).

PCA2 revealed a positive relationship between forest-floor moisture content, pH, N, P, and S concentrations, pri-mary nutrients for microbial growth, and Shannon’s diver-sity index. The negative relationship between Shannon’sdiversity index and the qCO2 value of the microbial com-munity suggests that a more diverse microbial communitymay be more efficient in terms of microbial C use.

The scatter plot of mature stands and the radiata pinechronosequence derived from the PCA is shown in Fig. 5b.The t tests of PCA1 scores obtained from forest floors indi-cated that PCA1 scores differed significantly between the Land FH layers (P < 0.05), with substantially higher scoresfor forest-floor samples from L layers. Furthermore, signifi-cantly lower PCA1 scores were obtained for the oak standthan for the beech and 40 year pine stands. Although thefirst factor was not able to differentiate the beech stand

Table 4. Substrates with high correlationcoefficients for PCA1 and PCA2 of metabolicdiversity patterns (CLPP) for mature standsand the radiata pine chronosequence.

rPCA1L-Asparagine 0.913

4-Hydroxy benzoic acid 0.797Glycyl-L-glutamic acid –0.605

D-Mannitol –0.817

PCA2D-Lactose 0.653

2-Hydroxy benzoic acid 0.826

D-Malic acid 0.838

Pyrubic acid methyl ester –0.713

Fig. 2. Microbial biomass (a), basal respiration rates (BR) (b), andmetabolic quotients (qCO2) (c) of microbial communities from theL (black bars) and FH (gray bars) layers from forest floors (FF). Adifferent lowercase letter indicates a significant difference betweenmature stands, and a different uppercase letter indicates a signifi-cant difference between stands in the radiata pine chronosequence.The bars represent means ± SD (n = 3).

1308 Can. J. For. Res. Vol. 39, 2009


from the 40 year pine stand, they showed significantly dif-ferent PCA2 scores. The FH layers of these stands alsoshowed significantly different PCA2 scores, with substan-tially higher scores for the beech stand than for the 40 yearpine stand. The forest floors from the 16 and 40 year pinestands were not significantly different in either forest-floorlayer.

Discussion

Effect of tree species on forest floorThe results indicate differences in detritus mass, which

may be at least partly due to differences in decompositionrates in the forest floors of the studied stands. Litter fromoak trees was so amenable to decay that it did not form a

Fig. 3. Cumulative respiration rates of the microbial community from the L (a) and FH (b) layers in the forest floor (FF) over 28 days ofincubation. Values represent means ± SD (n = 3).

Fig. 4. Shannon’s diversity index values for the L layers (solidbars) and FH layers (shaded bars) from forest floors. A differentlowercase letter indicates a significant difference between maturestands, and a different uppercase letter indicates a significant dif-ference between stands in the radiata pine chronosequence. Barsrepresent means ± SD (n = 3).

Fig. 5. Principal component analysis of a whole standardized dataset of forest floors from mature stands and the radiata pine chrono-sequence. (a) Loading factors plot. (b) Sample plot. Closed symbolsrepresent L-layer samples and open symbols FH-layer samples; cir-cles represent oak, triangles represent beech, squares represent 40year pine, and diamonds represent 16 year pine.



FH layer. These differences have been attributed to variousfactors, such as environmental factors (e.g., Dilly andMunch 1996), the quality of litter (Melillo et al. 1982), andthe activity of soil fauna (Irmler 1995; Priha et al. 2001).Raulund-Rasmussen and Vejre (1995) also observed veryrapid forest-floor decay under common oak in Danish for-ests and concluded that it was probably due to the activityof earthworms. On the other hand, beech stands showedhigher rates of forest-floor accumulation than pine stands,which may be due to the greater amount of litterfall pro-duced by beech (Kavvadias et al. 2001; Romanya and Val-lejo 2004). This is consistent with the findings of Kavvadiaset al. (2001), i.e., greater forest-floor accumulation underFagus sylvatica than under Pinus pinaster and Pinus nigra,but faster litter decomposition in beech than in pine forests.The decomposition rate of forest floor beneath pine ap-peared to be lowest, with 70% of the forest floor composedby the FH layer.

The L layer of the forest floor under deciduous trees hada different chemical composition from that of pine forestfloor. The pH and Ca concentration in the forest floor undertwo deciduous tree species (oak and beech) were signifi-cantly higher than in the forest floor under coniferous trees.Although bacterial activity predominates and nutrients arerapidly released from organic matter with a high pH andhigh concentrations of nutrients (Melillo et al. 1982), in thepresent study no differences were observed between broad-leaved and coniferous tree species in terms of MBC in theL layer. However, tree species can also influence microbialdecomposition primarily via differences in litter lignin con-tent (Hobbie et al. 2006). Litter with higher lignin/N and C/N ratios decomposes more slowly than litter with lowerlignin/N and C/N ratios (e.g., Melillo et al. 1982; Prescott1995; Hobbie et al. 2006). C/N and ADR/N ratios were sig-nificantly lower in deciduous trees than in pine trees. How-

ever, there were significant differences between the twodeciduous tree species in terms of forest-floor decomposi-tion rates, which may be explained by differences in the mi-crobial community.

The grouping of the CLPP samples according to the Eco-plates was not clear (Fig. 6), as was also reported by Prihaet al. (2001). Those authors showed that phospholipid fattyacid (PLFA) analysis was more sensitive to changes inmicrobial-community structure. This is probably due to thefact that PLFA analysis assesses the whole community,whereas CLPP, with Ecoplates, only measures the metabolicprofiles of culturable bacteria (Garland and Mills 1991).

The moisture content of the forest floor beneath beechtrees was higher than that of the forest floor beneath oaktrees, and as all samples were collected at the same time,these differences could be attributed to the higher water-storage capacity of the beech forest floor than the oak forestfloor, owing, for instance, to a higher accumulation of or-ganic matter. The difference in moisture content may influ-ence the bacteria:fungi ratio of the microbial community.Grayston and Prescott (2005) also concluded that the highbacteria:fungi ratio in cedar forest floors in British Columbiamay be related to the higher moisture content. Gram-negative bacteria, including Pseudomonas, are particularlysensitive to water stress, and Gram-positive bacteria, partic-ularly Actinobacteria, are highly tolerant of water stress(Killham 1994). The composition of the microbial com-munity and not the potential utilization of C sources maydiffer in the forest floor beneath oak and beech trees,which may explain the differences in organic-matterdynamics in the forest floor.

Effect of clear-cut forestry on the forest floorThe evidence for forest-floor loss following clear-cut and

mechanized operations was convincing and consistent withthe results of Johnson et al. (1995), who found a significantdecline in forest-floor mass 3–8 years after whole-tree har-vesting. However, some studies in which the same standswere compared before and after clear-cutting showed thatforest-floor mass increases when aboveground organic resi-dues are left in the stand (Mattson and Swank 1989). Theresults of the present study reflect the total removal of theforest floor from the 3 year pine stand with the front bladeof a bulldozer, and that it was still absent 3 years after theestablishment of the new pine plantation. Moreover, forest-floor mass was still 50% lower than in the mature stand16 years after disturbance. Covington’s curve (1981) pre-dicted a loss of 50% of forest-floor organic matter in thefirst 20 years after the disturbance, owing to a reduction inlitter input and acceleration of decomposition after clear-cutting. In the corresponding study, a series of stands ofdifferent ages were used to describe the pattern of forest-floor mass and organic-matter content during successionfollowing logging in northern hardwood stands in NewHampshire (Covington 1981).

Clear-cutting and subsequent site-preparation operationsmay also alter the quality of organic matter by increasingdecomposition rates. Ussiri and Johnson (2007) found rela-tively small changes in the structure and composition of or-ganic matter by 13C NMR after clear-cutting; however, theyconcluded that more labile fractions and more dynamic

Fig. 6. Principal component analysis of Ecoplate data obtainedfrom forest-floor samples of different layers in mature stands andthe radiata pine chronosequence incubated for 72 h. Closed symbolsrepresent L-layer samples and open symbols FH-layer samples; cir-cles represent oak, triangles represent beech, squares represent 40year pine, and diamonds represent 116 year pine. Values representmeans ± SD (n = 3).

1310 Can. J. For. Res. Vol. 39, 2009


pools of organic matter may be more sensitive indicators ofthe effects of disturbance. Water-soluble carbohydrates andcumulative respiration in the FH layer increased signifi-cantly relative to the adjacent mature pine forest in thestudied stand 16 years after clear-cutting. This may reflecthigher decomposition rates even 16 years after disturbance(Covington 1981).

The forest-floor microbial community was affected byclear-cutting and subsequent site preparation only in the FHlayer. The Shannon’s diversity index value was significantlylower than that observed in mature pine. Hannam et al.(2006) found that the forest-floor microbial community re-sponded immediately after harvesting but returned to pre-harvest PLFA levels 4.5 and 5.5 years postharvest. Incontrast, we suggest that although the CLPP were not af-fected by clear-cutting and subsequent site preparation, thediversity of the microbial community is not resilient follow-ing clear-cutting and subsequent site preparation during thefirst 16 years after disturbance of the FH layer.

ConclusionsForest floor under oak, beech, and mature pine stands var-

ied in terms of organic-matter dynamics. Differences amongtree species were found mainly in the L layer of the forestfloor. Functional microbial diversity was lower, microbialrespiration rates were higher, and the quality of litter waspoorer (high C/N and ADR/N ratios) in this layer in the ma-ture pine stand than in the deciduous stands. However,grouping of microbial communities according to CLPP wasnot clear, and the characteristics of the organic matter in thesoils, determined by CPMAS 13C NMR spectra, showedonly subtle differences among tree species.

Clear-cutting and mechanical site preparation in P. radi-ata stands cause a significant loss of organic matter, andthis may persist for at least 16 years after disturbance. In ad-dition, accelerated microbial respiration and substantiallylower microbial diversity were observed in the FH layerfrom the 16 year pine stand. However, further studies are re-quired to elucidate the effects of change in tree species andof clear-cutting and site preparation on C dynamics andlong-term site productivity.

AcknowledgementsFunding for this study was provided by the European

Union (ERDF-INTERREG IIIB Atlantic Area) and the De-partment of Agriculture, Fisheries and Food of the BasqueCountry. We are grateful to Dr. Fernando Blanco for helpwith the proximate analyses and Dr. Juan Mari Alberdi fortechnical assistance with NMR analysis. We also thank theUniversity of the Basque Country for supporting us withcomputer facilities and software packages, and Dr. ChristineFrancis for revising the English of the manuscript.

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Effects of tree species and clear-cut forestry on forest-floor characteristics in adjacent temperate forests in northern Spain