P stoichiometry in forest floor and A horizons as affected by tree species

P stoichiometry in forest floor and A horizons as affected by tree species

Soil Biology & Biochemistry 111 (2017) 166e175 Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier...

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Soil Biology & Biochemistry 111 (2017) 166e175

Contents lists available at ScienceDirect

Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio

Microbial biomass phosphorus and C/N/P stoichiometry in forest floor and A horizons as affected by tree species Dan Paul Zederer a, b, Ulrike Talkner b, Marie Spohn c, Rainer Georg Joergensen a, * a

Department of Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany €tzelstr. 2, 37079 Go €ttingen, Nutrient Management Group, Department of Environmental Control, Northwest German Forest Research Institute, Gra Germany c Department of Soil Ecology, University of Bayreuth, Dr.-Hans-Frisch-Str. 1-3, 95448 Bayreuth, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 November 2016 Received in revised form 27 March 2017 Accepted 15 April 2017

Forest floor horizons contain significant total P stocks, but information on the contribution of microbial biomass P (PMB) and on the controlling factors of this pool is limited. Slightly modified fumigation extraction procedures were used to investigate the stoichiometric relationships of PMB to microbial biomass C (CMB) and microbial biomass N (NMB) in the forest floor (L, F, H, and A horizons) at five sites, differing in P availability to trees, under adjacent spruce (Picea abies) and beech (Fagus sylvatica) stands. CMB, NMB, and PMB contents were higher in forest floors under beech than under spruce. Mean stocks of PMB and total P were roughly 27 and 100 kg ha1 in the forest floor, respectively, but did not differ between the tree species, due to an increased organic matter accumulation in the forest floor under spruce. This reveals the importance of forest floor horizons and microbial biomass turnover for P nutrition of trees in acidic soils with the humus form moder. C/PMB ratios declined from roughly 26 in L to 13 in F and H horizons, followed by an increase to roughly 17 in A horizons. The range of C/PMB ratios was small at all sites in relation to the wide SOC/total P ratios of the litter used as microbial substrate, indicating a relatively strict homeostatic regulation of the forest floor microbial, mainly fungal biomass stoichiometry. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Forest floor Fumigation extraction Bray-I extractable P Soil microbial biomass Spruce Beech

1. Introduction Forest floors are important P reservoirs in forest ecosystems. In the presence of moder-type humus, trees have been shown to take up considerably more phosphate from the forest floor than from the underlying mineral soil (Brandtberg et al., 2004; Jonard et al., 2009, 2010). When P-adsorbing mineral phases are lacking, the net release of plant available phosphate in forest floor horizons is determined by microbial mineralization and immobilization pro   cesses (Oberson and Joner, 2005; Rosling et al., 2016; Santr u ckova et al., 2004). Even under near steady state conditions, P is dynamically exchanged between microbial biomass and soil solution (Achat et al., 2010a, 2010c; Oehl et al., 2001). Consequently, the turnover of microbial biomass P (PMB) replenishes soil solution P and contributes to P nutrition of forest trees (Achat et al., 2012). The PMB pool in temperate mineral forest soil horizons has been

* Corresponding author. E-mail address: [email protected] (R.G. Joergensen). http://dx.doi.org/10.1016/j.soilbio.2017.04.009 0038-0717/© 2017 Elsevier Ltd. All rights reserved.

shown to be more closely correlated to soil organic C (SOC) or total N than to total P contents (Achat et al., 2012; Joergensen et al., 1995a,b; Khan and Joergensen, 2012). Achat et al. (2012) and Heuck et al. (2015) concluded that microbial P uptake is largely dependent on C availability, which determines the size of microbial biomass C (CMB). In the forest floor, where C availability to microorganisms is considerably higher than in mineral soils, as indicated by higher CMB/SOC ratios (Joergensen and Scheu, 1999a), P contents might influence PMB contents more strongly. However, the few results available from studies comparing PMB contents in forest floor horizons differing in total P contents are inconsistent. Saggar et al. (1998) found higher PMB contents in forest floors of P fertilized Pinus radiata stands in New Zealand than in non-fertilized stands. Clarholm (1993) observed the opposite in Swedish forest floors of P fertilized and non-fertilized Picea abies stands. C/PMB and also N/PMB ratios vary over a considerably wider vezrange than C/NMB ratios in temperate forest ecosystems (Cha Vergara et al., 2016; Joergensen et al., 1995a; Khan and Joergensen, 2012) as well as on a global scale (Cleveland and Liptzin, 2007; Hartman and Richardson, 2013; Xu et al., 2013).

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According to a modeling study by Manzoni et al. (2010), the high variability of C/PMB ratios is one of the main reasons for the limited power of litter C/P ratios in predicting net P release rates during litter decomposition. C/PMB ratios decrease under high P availability, combined with low C availability and high N availability in cell culture (Anderson and Domsch, 1980; Kouno et al., 1999; Lukito et al., 1998) and incubation studies (Salamanca et al., 2006). However, there is only limited knowledge on the impact of N and especially P availability on microbial C/N/P stoichiometry in temperate forest soil ecosystems (Joergensen et al., 1995a,b). This is especially true for forest floor horizons, where C, N, and P availability strongly depends on litter stoichiometry and decomposition stage (Mooshammer et al., 2014). Relationships between the elemental composition of forest floor horizons and microbial biomass have rarely been examined, due to methodological constraints (Joergensen and Scheu, 1999b; Saggar et al., 1998). For this reason, forest floor horizons have often been removed (Yang et al., 2010; Zhao et al., 2009) or have not been analyzed (Bing et al., 2016; Yang and Zhu, 2015) in studies on PMB in forest ecosystems. Microbial biomass contents and decomposition processes in forest floor horizons can be significantly affected by tree species. Different litter qualities of coniferous and deciduous species in respect to lignin and polyphenol contents, elemental composition, pH as well as physical characteristics (Berg and McClaugherty, 2014; Binkley and Giardina, 1998; Hobbie et al., 2007; Perry et al., 1987; Reich et al., 2005) have been reported to affect decomposition processes. CMB/SOC ratios have been found to be lower under coniferous than deciduous tree species (Bauhus et al., 1998; Scheu and Parkinson, 1995; Zhong and Makeschin, 2004). Although P contents of beech leaf and spruce needle litter might be similar (Hagen-Thorn et al., 2004; Trum et al., 2011), lower C and N availability to microorganisms in spruce compared with beech forest floors may also be associated with a lower microbial P demand, resulting in lower PMB contents. In contrast, the higher SOC stocks in spruce forest floors (Berger and Berger, 2012; Cremer et al., 2016; Vesterdal et al., 2013) may compensate for this and result in similar forest floor PMB stocks. In the present study, we investigated L, F, and H horizons as well as the upper A horizon of five adjacent beech (Fagus sylvatica L.) and spruce (Picea abies L.) stands at five sites, varying strongly in parent material and P availability. After conducting a methodological prestudy to test the effect of the type of extractant and of the soil/ extractant ratio on PMB estimates (Bergkemper et al., 2016; Brookes et al., 1982; Khan and Joergensen, 2012), we adapted the fumigation extraction method to measure PMB in forest floor horizons. Our central objectives were to investigate the following hypotheses: (I) CMB and PMB contents are higher in forest floors under beech than under spruce, but there is no tree species effect on C/PMB ratios. (II) Stocks of PMB in the forest floor do not differ between tree species, due to an increased forest floor accumulation under spruce. (IIIa) PMB contents are affected by total P in forest floor horizons and (IIIb) by SOC contents in A horizons. (IV) C/PMB ratios decrease with depth from L to A horizons as the substrate is increasingly degraded. 2. Materials and methods 2.1. Study sites, soil sampling and sample preparation Five paired forest sites, consisting of adjacent mature beech (Fagus sylvatica L.) and spruce (Picea abies L.) stands, were selected in Central and Northern Germany (Table 1). The beech stands at Vessertal and Goettingen as well as the beech and spruce stands at Solling are part of the International Co-operative Programme Forest Level II Intensive Monitoring Programme (Haussmann and Lux,

167

1997). The spruce stands at Vessertal and Goettingen are located 150 and 400 m apart from the respective beech plots. The beech and spruce stands at Oerrel are located on managed, non-fertilized control plots of the nutrient deficiency trial Oerrel-Lintzel, which was established in 1929 on formerly afforested heathland (Seibt et al., 1968). The beech and spruce stands at Meissner are located in the nature reserve park (Nitsche et al., 2005). Beech or spruce stands consisted of at least 90% beech or spruce trees, respectively, except for the Oerrel beech stand, which comprised 75% beech and 25% Scots pine trees. The soils of the five sites had formed from different parent materials (Table 1) and were chosen since they represent a P availability gradient. Four of the sites were selected based on foliar P contents of the beech stands in 2013 as a measure for P availability to trees, which decreased in the order Vessertal (1.6 mg g1 DM) > Solling (1.2 mg g1 DM) > Oerrel (1.0 mg g1 DM)  Goettingen (1.0 mg g1 DM). The Meissner site was selected due to its markedly high total P content of 1.8 mg g1 soil at 0e10 cm in the A horizon, but foliar P contents were not measured (Khan and Joergensen, 2012). Samples were taken in December 2013. At each site and stand, four sampling points were randomly selected within a 20 m  50 m rectangular area. At each sampling point, two (for the moder profiles) to four (for the mull profile) replicate soil cores with a diameter of 8 cm were taken, using a bi-partite root auger (Eijkelkamp, Giesbeek, Netherlands). Forest floor horizons were then differentiated into intact and mainly intact litter (L horizon), moderately to strongly fragmented and fermented litter (F horizon) and, if existing, humified material without any visible litter remains (H horizon). The separated organic horizon material of each core was collected quantitatively completely to enable the calculation of stocks on a mass-per-area basis (Raubuch and Beese, 1995). The upper 5 cm of the remaining mineral soil core were separated and denominated as A horizon. Samples of each sampling point were horizon-specifically bulked and stored in polyethylene bags at 4  C for a further 2 weeks. Prior to all analyses, L horizon material was cut with scissors to pass a 4-mm sieve. F material was passed through a 4-mm sieve without prior cutting, H and A material through a 2-mm sieve. For determining the water content and for chemical analysis (except for organic P), forest floor subsamples were dried at 80  C and A subsamples at 105  C for 24 h, respectively. 2.2. Microbial biomass analyses CMB and microbial biomass N (NMB) were estimated by fumigation extraction (Brookes et al., 1985; Vance et al., 1987). Field moist A horizon subsamples were split into two aliquots, equivalent to 7.5 g DM each, field moist forest floor subsamples were split into two aliquots, equivalent to 2 g DM each. One aliquot each was fumigated for 24 h at 25  C with ethanol-free CHCl3. Fumigated and non-fumigated A horizon samples were extracted with 45 ml 0.5 M K2SO4 by 30 min horizontal shaking at 200 rev min1 and filtered (3 €ttingen, Germany). Forest floor hw, Sartorius Stedim Biotech, Go samples were similarly extracted with 60 ml 0.5 M K2SO4, corresponding to a 1 g DM to 30 ml ratio (Stockfisch et al., 1995). Organic C and total N concentrations in the extracts were measured after combustion, using a multi N/C 2100 automatic analyzer (Analytik Jena, Jena, Germany). CMB was calculated as EC/kEC, where EC ¼ (organic C extracted from fumigated samples) - (organic C extracted from non-fumigated samples) and kEC ¼ 0.45 (Wu et al., 1990). NMB was calculated as EN/kEN, where EN¼ (total N extracted from fumigated samples) - (total N extracted from non-fumigated samples) and kEN ¼ 0.54 (Brookes et al., 1985). PMB was also determined by fumigation extraction (Brookes

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Table 1 Site names, substrate, elevation, mean annual precipitation, mean annual temperature, beech and spruce stand ages, soil type (FAO-WRB, 2014), texture, soil pH in the A horizon at 0e5 cm depth and forest floor horizon depths at the five experimental sites. Site

Meissner Vessertal Solling Oerrel Goettingen a e

Elevation

Precip.

Temp.

(m)

(mm)

( C)

b

b

b

700e720 800e850a 500e550a 100e150ac 400e450a

915 1446a 1088a 730ac 680a

7.4 5.3a 6.5a 8.0ac 7.8a

Substrate

Soil type

Clay

Silt

Sand

(%) Basalt Trachyandesite Loess loam Glacial sand Shell-lime

Eutric Andosol Eutric Cambisol Dystric Cambisol Aric Albic Podzol Rendzic Leptosol

19 19 21 6 56

73 52 60 24 39

9 29 19 70 5

Soil pH-H2O

Horizon depth (cm L/F/H)

Stand age (years)

Beech

Beech

Beech

3.9 3.9 3.5 3.6 6.0

Spruce 3.8 3.6 3.3 3.4 3.9

0.5/4.0/3.5 0.5/2.0/1.3 0.5/3.7/1.5 0.5/3.5/2.2 1.0/0.5/NA

Spruce 0.7/3.7/2.5 0.4/2.9/1.3 0.5/3.5/2.5 0.4/3.9/2.2 0.5/2.1/2.8

b

140 120b 140b 80d 130b

Spruce 70b 110e 130b 80d 110e

data taken from Haussmann and Lux (1997); b data taken from local forestry authorities; c data refer to the nearby Level-II site Luess; d data taken from Seibt et al. (1968); estimated values; NA ¼ not applicable.

et al., 1982). Preliminary tests were conducted to check whether Olsen extraction (0.5 M NaHCO3, pH 8.5) and Bray-I extraction (0.03 M NH4F þ 0.025 M HCl, pH 2.55) at different soil/extractant ratios (1/50, 1/100, 1/150, 1/200) give similar PMB values for forest floor horizons. Neither extractant type nor soil to extractant ratio affected PMB estimates significantly. In the final method, a sample of field moist A horizon material was split into three aliquots equivalent to 4.5 g DM each, samples of field moist L, F and H materials were split into two aliquots equivalent to 0.9 g DM each, respectively. One aliquot each remained untreated; each second aliquot was fumigated for 24 h at 25  C with ethanol-free CHCl3. The third aliquot of A horizon material was spiked with 25 mg P g1 soil added as KH2PO4 solution (250 mg P l1). All fumigated and non-fumigated aliquots were extracted with 90 ml Bray-I solution by horizontal shaking at 150 rev min1 for 30 min. This corresponded to a soil to extractant ratio of 1 g to 20 ml for A horizons (Khan and Joergensen, 2012) and 1 g to 100 ml for forest floor horizons. Subsequently, all suspensions were centrifuged at 2000 g for 10 min and filtered (3 hw, Sartorius € ttingen, Germany). PO4-P concentrations of the Stedim Biotech, Go extracts were measured photometrically according to a modified molybdate-ascorbic acid method (Olsen and Sommers, 1982) at 882 nm using a microtiter plate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). PMB was calculated as EP/kEP/R, where EP ¼ (PO4-P extracted from fumigated sample) - (PO4-P extracted from non-fumigated sample) and kEP ¼ 0.40 (Brookes et al., 1982). To correct for P sorption during the extraction of A horizon material, this value was divided by the recovery rate R of added P (25 mg PO4-P g1 soil): ((PO4-P extracted from spiked sample) - (PO4-P extracted from non-fumigated sample))/25. PMB contents of L, F and H horizons were not corrected for P sorption during extraction, because preliminary tests revealed P recovery rates of 96% for F and H horizons at all sites. Finally, it was also tested whether measuring total P in Bray-I extracts of fumigated and non-fumigated samples results in similar PMB estimates in comparison with those obtained by PO4-P measurement. For this reason, total P was additionally measured by ICP-OES (Vista-Pro radial, Varian, Palo Alto, USA) in Bray-I extracts of fumigated and non-fumigated samples of the Solling site. PMB contents were calculated as described above, except for using total P concentrations instead of PO4-P concentrations. For the A horizon, PMB contents calculated from the total P concentrations of the extracts (TP-PMB) and those calculated from the PO4-P concentrations of the extracts (PO4-PMB) were highly correlated (R2 ¼ 0.99, Appendix-Fig. 1a) and did not differ significantly (paired t-test, p ¼ 0.12). For forest floor horizons, TP-PMB contents and PO4-PMB contents were also closely correlated (R2 ¼ 0.98, Appendix-Fig. 1b), but PO4-PMB significantly exceeded TP-PMB by 10% (paired t-test, p < 0.001). However, regarding the high variability of measured kEP values (e.g. Brookes et al., 1982), these differences are still negligible.

2.3. Chemical analysis Soil pH was measured in a soil to solution ratio of 1 g to 10 ml water for forest floor horizons and 1 g to 2.5 ml water for A horizons (Stockfisch et al., 1995). Total C and N were determined by gas chromatography after dry combustion using a Vario EL (Elementar, Hanau, Germany) analyzer. Total C was considered as organic C because no sample contained carbonate. Soil texture was determined by wet-sieving (sand fraction > 63 mm) and gravitational sedimentation (silt and clay fraction) after removing organic materials with 10% H2O2 (Blume et al., 2011). Total P of all horizons was extracted with aqua regia. Briefly, 0.2 g of finely ground, oven dry material was weighed into HClwashed Teflon flasks, moistened with 0.5 ml ultra-purified water and subsequently mixed with 6 ml HCl (32%) and 2 ml HNO3 (65%). After a pre-reaction time of 30 min, samples were put into a microwave (mPREP-A, MLS, Leutkirch, Germany), heated up to 220  C for 10 min and subsequently pressure-digested at 220  C for 20 min. When cooled to 40  C, the contents of the Teflon flasks were quantitatively transferred to 50 ml volumetric flasks and made up to 50 ml using ultra-purified water. Finally, extracts were shaken up, filtered through an acid resistant filter paper (Whatman 40) and stored at 4  C until measurement. Total P concentrations of the extracts were measured by ICP-AES at 213.618 nm wavelength (Vista RL CCD Simultaneous, Varian, Palo Alto, USA). The organic P content of A horizons was determined by an ignition method (Saunders and Williams, 1955; Walker and Adams, 1958) as described by Olsen and Sommers (1982). A sample of airdried soil was divided into two 1-g aliquots, one of them being placed in a porcelain crucible, the other in a polypropylene centrifugation tube. The aliquot in the porcelain crucible was ignited at 550  C for 1 h and then also transferred to a polypropylene tube. Subsequently, both aliquots were extracted with 50 ml 0.5 M H2SO4 at 150 rev min1 for 16 h, centrifuged at 1500 g for 15 min and filtered through an acid resistant filter paper (Whatman 40). Extracts were adjusted to pH 1.5 using 4 M NaOH. PO4-P concentrations of the extracts were measured as described for PMB extracts. The organic P content was calculated by subtracting the PO4-P content of the non-ignited sample from the PO4P content of the ignited sample. 2.4. Statistical analysis The results presented are arithmetic means and expressed on an oven-dry basis. All elemental ratios are presented on a mass basis. For each parameter and horizon, the mean values of each of the five sites were used for a statistical evaluation of the tree species effect. Therefore, an analysis of variance was performed with a linear mixed-effects model fitted by restricted maximum likelihood (REML), with “tree species” being included as a fixed effect and “site” as a random effect. Normal distribution of the residuals was

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Table 2 SOC, total N, and total P contents as well as SOC/total N and SOC/total P ratios for the forest floor horizons and the upper 5 cm of the A horizon of beech and spruce stands at five sites. Horizon

SOC (mg g1 soil)

Total N (mg g1)

Total P (mg g1)

Beech

Spruce

Beech

Spruce

Beech

500 470 350 120

12.3 22.2 17.5 5.7

18.2 20.4 18.2 6.8

0.70 1.63 1.96 1.64

1.20 1.10 1.97 2.09

42 18 17 15

28 23 19 17

740 250 150 50

420 430 180 56

490 440 260 62

11.9 22.8 15.5 3.9

15.2 19.2 13.9 3.2

1.13 1.67 1.70 1.08

1.12 1.22 1.24 0.49

43 20 18 17

32 23 19 19

450 270 160 60

440 370 210 126

510 500 440 74

12.3 24.1 20.6 2.9

18.7 20.8 18.1 3.6

0.72 1.04 1.41 0.56

1.09 1.00 1.16 0.48

43 21 19 18

27 24 24 21

730 490 280 94

470 510 380 160

510 500 430 52

9.0 20.7 15.8 2.3

16.7 19.3 15.2 2.0

0.39 0.89 0.74 0.18

0.71 0.85 0.75 0.17

57 24 25 24

31 26 28 26

1330 570 520 310

730 590 570 320

490 390 340 58

11.1 18.1 NA 6.4

11.6 16.9 15.7 3.6

0.47 1.09 NA 1.06

0.65 1.14 1.18 0.61

45 22 NA 13

42 23 22 16

1080 360 NA 77

760 340 290 100

11.3* 21.6* 17.4 4.2 7.7

16.1* 19.3* 16.2 3.8 9.0

0.95* 1.06 1.26 0.77 9.0

46* 21* 20* 17* 4.4

32* 24* 22* 20* 5.1

870 390 280 120 11

560 450 330 150 10

Meissner L 520 F 400 H 290 A 83 Vessertal L 510 F 440 H 270 A 64 Solling L 520 F 500 H 390 A 52 Oerrel L 520 F 500 H 390 A 56 Goettingen L 500 F 390 H NA A 81 Mean over all sites L 513* F 447 H 337 A 67 CV (±%) 7.3

501* 460 362 73 7.5

0.68* 1.26 1.46 0.90 8.2

SOC/total N Spruce

Beech

SOC/total P Spruce

Beech

Spruce

CV ¼ pooled coefficient of variation between replicate samples (n ¼ 4 for each stand and horizon); NA ¼ not applicable; * indicate a significant difference between tree species across all sites (P < 0.05).

investigated by visual inspection of the normal probability plots. Differences between horizons were not tested statistically. Pearson's product moment correlations and regression analyses were calculated separately for the L horizon, the grouped F þ H horizons, and the A horizon to account for the strong horizon-specific clustering of some parameters. Multiple linear regression models were calculated for the PMB content as a dependent variable. Independent variables were selected stepwise (bidirectional) by Akaike's Information Criterion (AIC) from the following variables: CMB contents, NMB contents, pH, SOC contents, total N contents, total P contents and in case of A horizons organic P contents. The residuals of all models were tested for normal distribution (Shapiro-Wilk test and visual inspection of the normal probability plots), homoscedasticity (Breusch-Pagan test) and absence of autocorrelation (Breusch-Godfrey test). Furthermore, variance inflation factors (VIF) were calculated for the final models to test for the absence of multicollinearity, considering a VIF of 4 as threshold for all predictors. The significance level for all tests was set at P < 0.05. All statistical analyses were carried out using R 3.0.2 (R Core Team, 2013), and the nlme-package (Pinheiro et al., 2012) was used for the linear mixed-effects model. 3. Results

horizons, 4.5 ± 0.8 SD in F horizons, to 3.9 ± 0.2 SD in H horizons, without significant tree-species effects. SOC contents decreased from an overall mean of 507 in L to 70 mg g1 soil in A horizons (Table 2), with little difference between tree species. Total N increased from L to F horizons, followed by a decrease from F to A horizons almost proportionally to SOC. Consequently, the SOC/total N ratios varied around 19 under beech and around 22 under spruce in these 3 horizons. Total P contents generally increased from L to H horizons. Total P contents were significantly lower in the beech L horizons, consisting of freshly fallen leaf litter, than in spruce L horizons, consisting of aged needle litter. Total P contents of all other horizons were similar across both tree species. In F and H horizons, standspecific total P contents varied between 0.74 (Oerrel beech) and 1.97 mg g1 (Meissner spruce). In A horizons, stand-specific total P contents varied much more strongly between 0.17 (Oerrel spruce) and 2.09 mg g1 soil (Meissner spruce). SOC/total P ratios consistently declined from a maximum of 1330 in L (Oerrel beech) to a minimum of 50 in A horizons (Meissner beech). On average across all sites and horizons, 3.0% ± 2.1% SD of total P was P-Bray-I extractable, without significant tree-species effects, due the high variation of 29% between replicate sampling points. Organic P contributed on average 63% to total P of A horizons, without significant tree-species effects (Fig. 1).

3.1. Soil chemical properties 3.2. Soil microbial biomass indices The A horizons were strongly acidic at all sites, the only exception being the Goettingen beech stand (pH 6.0), which is due to the calcareous bedrock (Table 1). Forest floor pH decreased with depth at all sites from 5.2 ± 0.7 SD (standard deviation) in L

CMB contents strongly declined from roughly 13.5 in L to a mean of 0.81 mg g1 soil in A horizons (Table 3). CMB contents of beech F and H horizons significantly exceeded those of the respective

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F þ H and A horizons than in L horizons. The linear relationships between PMB and NMB contents were even stronger than those to CMB contents. PMB and total P contents were closely correlated in beech L horizons, but less closely in spruce L horizons than in F þ H and A horizons across both species (Fig. 4b, Table 5). In spruce L horizons, the relationship of PMB and total P was increased by pH and NMB (Table 5). In A horizons, PMB contents were not related to total P contents, but were affected by the combined effects of NMB and clay contents. 4. Discussion 4.1. Tree species effects PMB contents were significantly lower under spruce than under beech in all forest floor horizons. No clear indications were found Fig. 1. Organic P as percentage of total P in the upper 5 cm of the A horizon of beech and spruce stands at five sites.

spruce horizons by 54 and 24%. Depth gradients and tree-species effects on CMB were also reflected by the CMB/SOC ratios. NMB was highly correlated with CMB in F þ H (r ¼ 0.99) and A horizons (r ¼ 0.93), but somewhat lower in L horizons (r ¼ 0.69). The C/NMB ratios varied around 10.2 in L horizons across both species and declined to an overall mean of 7.4 in spruce F, H, and A horizons, which exceeded that in the respective beech horizons by 14%. PMB contents consistently declined with depth (Fig. 2aee) from a maximum of 1330 in L (Vessertal beech, Fig. 2b) to a minimum of 29 mg g1 soil in A horizons (Oerrel beech and spruce, Fig. 2d). In F and H horizons, stand-specific means of PMB varied between 179 (Oerrel spruce) and 782 mg g1 soil (Vessertal beech). Across all horizons, the mean PMB contents under spruce were roughly 35% lower than under beech (Fig. 2aee, Table 4). This difference was significant in all forest floor but not in the A horizons. Across both tree species, C/PMB ratios declined from roughly 26 in L to 13 in F and H horizons, followed by an increase to 17 in A horizons. C/PMB ratios were not related to C/NMB ratios, but showed a significant non-linear relationship to the P-Bray-I contents (Fig. 3). The contribution of PMB to total P also declined with depth (Fig. 2 fej) from a maximum of 116% in L (Meissner beech, Fig. 2f) to a minimum of 1.6% in A horizons (Meissner spruce, Fig. 2f). In L and F horizons, mean PMB/total P ratios of spruce stands were roughly 65% lower than those of beech stands, but similar in H and A horizons (Fig. 2fej, Table 4). Mean PMB stocks were about 27 kg ha1 in the forest floor, but only 18 kg ha1 in A horizons at the upper 5 cm, which is equivalent to 27% and 7% of the total P stocks, respectively. Across both tree species, PMB contents were significantly correlated with CMB (Fig. 4a, Table 5). This was more pronounced in

Table 3 CMB and NMB contents as well as C/NMB and CMB/SOC ratios for the forest floor horizons and the upper 5 cm of the A horizon of beech and spruce stands, mean over all five sites. Horizon CMB (mg g1 soil) NMB (mg g1 soil) C/NMB

CMB/SOC (%)

Beech

Spruce

Beech

Spruce

Beech Spruce Beech Spruce

L 13.60 F 6.93* H 4.23* A 0.98 CV (±%) 16

13.50 4.51* 3.40* 0.64 14

1.40 1.18* 0.57* 0.18 17

1.28 0.72* 0.41* 0.09 17

9.8 6.0* 7.5* 6.1* 6.8

10.5 6.4* 8.6* 7.3* 7.9

2.65 2.69 1.59* 1.00* 1.33* 0.99* 1.39 0.89 16 13

CV ¼ pooled coefficient of variation between replicate samples (n ¼ 4 for each stand and horizon); * indicate a significant difference between tree species across all sites (P < 0.05).

Fig. 2. (a, b, c, d, e) PMB contents and (f, g, h, i, j) PMB/total P ratios for the forest floor horizons and the upper 5 cm of the A horizon of beech and spruce stands at five sites; error bars indicate ± one standard error of mean for replicate samples (n ¼ 4).

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Table 4 PMP contents, C/PMB and PMB/total P ratios, stocks of PMB and total P for the forest floor horizons and the upper 5 cm of the A horizon of beech and spruce stands, mean over all five sites. Horizon

L F H A CV (±%)

PMB (mg g1 soil)

C/PMB

PMB (kg ha1)

Beech

Spruce

Beech

Spruce

Beech

Spruce

Beech

Spruce

Beech

Spruce

640* 540* 390* 75 18

460* 310* 280* 39 18

23 13 12 15 11

29 15 13 18 11

95* 42* 28 10 17

50* 30* 23 9 15

3 13 14 22 27

2 13 12 13 24

3 32 51 273 21

4 41 59 226 21

PMB/total P (%)

Total P (kg ha1)

CV ¼ pooled coefficient of variation between replicate samples (n ¼ 4 for each stand and horizon); * indicate a significant difference between tree species across all sites (P < 0.05).

that this was related to bioavailability of P in spruce forest floors. Total P contents were significantly lower in L horizons only and PBray-I contents did not differ significantly in any horizon. There were also no tree-specific differences in accumulation of organic P components in A horizons. Especially in F and H horizons, tree species effects on PMB were similar to those on CMB contents, resulting in non-significant differences between the C/PMB ratios, which seems to support our hypothesis I. However, C/PMB ratios were consistently 8 to 26% larger in all horizons of the spruce stands, indicating the possibility of stronger substrate quality effects on PMB than on CMB contents. Although C/PMB and C/NMB ratios were not correlated, they were also consistently 7 to 20% larger in all horizons of the spruce stands, but this difference was in most cases significant due to lower spatial variation between replicate sampling points. One reason may be differences in the microbial community structure, especially differences in tree-specific ectomycorrhizal fungi (Goldmann et al., 2015). Contents of CMB and NMB, C/NMB ratios as well as CMB/SOC and NMB/total N ratios were in a range typical for forest floor horizons of temperate deciduous and coniferous forests (Joergensen and Scheu, 1999a; Raubuch and Beese, 2005; Scholle et al., 1992; Stockfisch et al., 1995; Zhong and Makeschin, 2004). In F and H horizons, CMB and NMB contents were lower under spruce than under beech, which is generally in agreement with results from Thuringia (Zhong and Makeschin, 2004) and the Solling (Bagherzadeh et al., 2008). Also the CMB/SOC and NMB/total N ratios, indicators for substrate availability to soil microorganisms (Anderson and Domsch, 1989; Dilly et al., 2003; Joergensen and Emmerling, 2006), were lower under spruce than under beech.

Fig. 3. Non-linear (3 parameter exponential decay: y ¼ 10.8 þ 19.9(0.044x), r ¼ 0.56; P < 0.0001) relationship between C/PMP ratios and P-Bray-I contents of pooled beech and spruce forest floor horizons of five sites.

A lower substrate availability of spruce needle litter compared with beech leaf litter to soil microorganisms may be related to a lower N availability, as indicated by higher SOC/total N ratios in forest floor horizons under spruce compared with beech. Additionally, spruce litter likely contains more phenolic components than beech litter (Kuiters and Denneman, 1987), which reduce C €ttenschwiler and and N availability to soil microorganisms (Ha Vitousek, 2000; Kuiters, 1990).

Fig. 4. (a) Linear relationships between CMB and PMB contents, calculated separately for L, F þ H and A horizons (L: r ¼ 0.44, F þ H: r ¼ 0.86, A: r ¼ 0.81, p < 0.01 in all cases). (b) Relationships between total P and PMB contents, calculated separately for beech L, for spruce L, and for both species F þ H and A horizons (L beech: r ¼ 0.93, L spruce: r ¼ 0.60, F þ H: r ¼ 0.54, A: r ¼ 0.26, p < 0.01 in all cases, except for the A horizon, where p ¼ 0.10).

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Table 5 Multiple linear regression models for the PMB as a dependent variable, calculated separately for L, F + H and A horizons; independent variables were selected in a stepwise (bidirectional) procedure from the following parameters: CMB, NMB, pH, SOC, total N, total P, and for A horizons also organic P; parameter estimates shown are standardized (b) coefficients. Dependent variable: PMB Horizon

Species

Constant

Independent variables

Adjusted R2

SEE

L L

Beech Spruce

28 716**

0.86 0.70

95.3y 51.5

F+H

Beech

0.89

58.7

F+H

Spruce

0.82

33.7x

A

Beech

0.96

11.3

A

Spruce

0.93 0.94 0.71 0.40 0.95 0.35 0.31 0.83 0.36 0.85 0.15 1.20 0.98

0.43

9.5

83

61* 2 109*

total P*** total P** pH** NMB* CMB*** total P*** pH*** CMB*** total P*** NMB*** clay* clay*** NMB***

difference. Another reason might be that the kEP value of 0.40 is too low for converting CHCl3-labile P into PMB during early stages of decomposition, although this value is only a little below the 0.45 proposed by Achat et al. (2009, 2010b), based on an isotope dilution approach. Fungi release more CHCl3-labile material than bacteria in pure cultures (Brookes et al., 1982; Eberhardt et al., 1996; Greenfield, 1995), but an effect of the fungal/bacterial biomass ratio has not been proven for real soils. A kEP factor must be used to correct for the non-extractable part after fumigation, even in the absence of P fixing soil colloids. However, the extraction of an additional P-spiked sample is not necessary in this case. Enzymatic hydrolysis of non-biomass organic P during fumigation (Achat et al., 2010b; Morel et al., 1996) would require the leakage of phosphatases from microbial cells during fumigation, which is unlikely (Brookes et al., 1982). Some phosphatases might be released during extraction (Noack et al., 2012), but the pH of the Bray-I extractant is too low and the extraction period is too short to allow for substantial enzymatic hydrolysis. 4.3. Forest floor specific effects on PMB contents and C/PMB ratios

Across all sites, average forest floor SOC stocks under spruce exceeded those under beech by 45%, confirming the stronger SOC accumulation in spruce forest floors (Berger and Berger, 2012; Cremer et al., 2016; Vesterdal et al., 2013). However, this did not significantly affect total P stocks in spruce compared with beech forest floors, as observed by Vesterdal and Raulund-Rasmussen (1998). Mean PMB stocks were roughly 27 kg ha1 in beech and spruce forest floors, supporting our hypothesis II. Forest floor PMB stocks were not only similar between tree species but also between most of the investigated sites, which is remarkable, considering the strong differences in total P contents and stocks in the mineral soil. Even at the Oerrel site, a sandy Podzol, which exhibited lowest P contents in our study, forest floor PMB stocks amounted to 24 kg ha1 across beech and spruce stands. Assuming a PMB turnover time of 0.5e1.0 year (Achat et al., 2010b; Chen et al., 2003; Oehl et al., 2001), an average P flux of 24 to 48 kg P ha1 a1 through the microbial biomass could be calculated for the forest floors of this site. From this turnover, sufficient P will most likely be diverted to maintain tree requirements of roughly 5 kg P ha1 a1 (Ellenberg et al., 1986) in temperate beech and spruce stands. This reveals the importance of forest floor horizons and the microbial biomass turnover for P nutrition of trees in acidic soils with the humus form moder (Jonard et al., 2010, 2009). 4.2. PMB estimations in forest floor horizons The contribution of PMB to total P averaged over all forest floor horizons under spruce ranged from 16 to 35%, which is in the range reported for forest floors of northern spruce stands (Christ et al., 1997; Clarholm, 1993). Similarly, PMB contributed 25 to 35% to total P in the organic layers of arctic heathlands (Jonasson et al., 1996, 1999; Schmidt et al., 1999, 2002). In beech forest floors, this percentage ranged slightly higher from 22 to 47%, which is still below the maximum values of 53 to 73% reported for forest floors of different temperate pine stands (Achat et al., 2010c; Chen et al., 2003; Saggar et al., 1998). Evidence for the high contribution of PMB to total P in forest floor horizons is not restricted to results obtained by the CFE method. In a 1H and 31P NMR correlation study, Vincent et al. (2013) found that on average 40% of the organic P in boreal forest floors was phospholipid- and RNA-P, most likely derived from the microbial biomass. In some beech L horizons, PMB contents slightly exceeded total P contents. One reason might be simply error accumulation of P measurements of fumigated and non-fumigated samples on the

In L horizons, PMB and CMB contents were closely related to total P (beech: r ¼ 0.60, spruce: r ¼ 0.75, P < 0.01), suggesting P effects on microbial biomass development. A sole relationship between PMB and total P may be caused by luxury P consumption of microorganisms with increasing P availability (Scott et al., 2012). However, this does not explain the relationship between CMB and total P contents. Similarly strong positive relationships between CMB and total P have been observed for peat soils, which are very low in total P (Brake et al., 1999), but not for mineral A horizons on a global scale (Hartman and Richardson, 2013). In NaH2PO4 amended plots of the Goettingen beech stand, Joergensen and Scheu (1999b) observed not only a significant increase in PMB but also in CMB contents in L horizons. In F and H horizons, PMB contents were closely related to CMB and to a lesser extent to total P contents. This suggests that microbial P incorporation was predominantly dependent on the CMB pool (Achat et al., 2012). Consequently, there was no significant correlation between CMB and total P contents and no indication of microbial P limitation in F and H horizons. In line with this, a higher P availability compared with the L horizons was indicated by lower SOC/total P ratios as well as by higher P-Bray-I contents. However, no general convergence of SOC/total P ratios was observed with increasing degree of decomposition from L to H horizons as proposed by Liu et al. (2016). Although total P affected PMB in all forest floor horizons, it was obviously not the main driver of PMB contents in F and H horizons, only partly supporting our hypothesis IIIa. In these two horizons with SOC/total P ratios of up to 592, P availability was apparently high enough to meet the physiological demands of microorganisms at all sites and across tree species. Saggar et al. (1998) estimated a threshold SOC/total P ratio of about 550 for net P mineralization in forest floor samples of New Zealand pine (Pinus radiata) plantations. A recent study found that net P mineralization only occurred below C/P ratios of 1400 and N/P ratios of 40 in slightly decomposed forest floor horizons of temperate forests in Germany (Heuck and Spohn, 2016), indicating that below this threshold microorganisms are not lacking P. Strong relationships between PMB and total P contents are only to be expected when this threshold is considerably exceeded, as in the current L horizons. In contrast, a pronounced relationship between PMB contents and SOC/total P ratios over the entire forest floor was reported for 43 French pine (Pinus pinaster) stands, with SOC/total P ratios of up to 1600 (Achat et al., 2012). The horizon specific changes in the relationship between CMB

D.P. Zederer et al. / Soil Biology & Biochemistry 111 (2017) 166e175

and PMB contents were also reflected in the C/PMB ratio. In L horizons, the C/PMB ratio ranged from 15 to 39 across the different sites and both tree species, which might be caused by differences in the vez-Vergara et al., 2016). fungal colonization after litter fall (Cha Although C/PMB ratios were not as strictly homeostatic as C/NMB ratios, the range of C/PMB ratios was astonishingly small at all sites in relation to the wide SOC/total P ratios of the litter used as mivez-Vergara et al. (2016) crobial substrate. In contrast, Cha measured C/PMB ratios of up to 65 in the L horizon of a Mexican Quercus deserticola stand. In the F and H horizons of the present study, the C/PMB ratios converged to a considerably smaller range from 10 to 17 across all sites and both tree species, which may also indicate luxury uptake. In contrast, values reported for F and H horizons by others are generally higher, ranging from 16 to 64 (Chen et al., 2003; Kandeler et al., 1999; Saggar et al., 1998; Sparling et al., 1994). The decline in C/PMB ratios with increasing decomposition from L to H horizons partly supports our hypothesis IV. The changes in the C/PMB ratio along the decomposition gradient from L, F to H horizons may be related to changes in the microbial community or to an adjusted P uptake of a rather uniform microbial community (Mooshammer et al., 2014). The latter explanation might apply if soil microorganisms take up P in excess of their physiological demand under high P availability, as has been shown for cultured fungi and bacteria (Koukol et al., 2006; Scott et al., 2012). If this were the case in soils, strict homeostasis of C/ PMB ratios would be expected only under P limitation (Hartman and Richardson, 2013). However, in the current study, a non-linear relationship between C/PMB ratios and P-Bray-I in forest floor horizons indicates that C/PMB ratios were much more variable under low than under high P availability. In this case, the abrupt increase of C/PMB ratios at low P availability may indicate the existence of a stoichiometric threshold that marks the shift from a microbial community not adapted to P limitation to one adapted to P limitation. In reference to Cleveland and Liptzin (2007), such a threshold may even mark the transition from non-P-limited to Plimited micro-ecosystems. To test this hypothesis, it would be necessary to relate C/PMB ratios to P mineralization and immobilization rates (Bünemann, 2015; Bünemann et al., 2016) and to mivez-Vergara et al., crobial community structural parameters (Cha 2016; Rosling et al., 2016; Zavisi c et al., 2016). 4.4. A horizon specific effects on PMB contents and C/PMB ratios In A horizons, PMB showed close relationships with CMB and NMB but not with total P, P-Bray-I or SOC, irrespective of the tree species. No evidence was found that PMB contents in mineral A horizons were controlled by C and P availability as previously suggested (Achat et al., 2012; Joergensen et al., 1995b; Khan and Joergensen, 2012). This contrasts our hypothesis IIIb. One reason for this discrepancy may be the strong effects of contrasting mineralogy on soil microorganisms in A horizons, despite the dominance of organic P at all sites. This assumption is supported by the effect of clay contents on the relationship between PMB and NMB. The inter-site variability of C/PMB ratios in A horizons was higher than in F and H horizons and similar to L horizons. Except for the Goettingen site, C/PMB ratios in A horizons were similar to or even higher than the overlying H horizon, which partly disagrees with our hypothesis IV. Additionally, this contrasts findings of Sparling et al. (1994) and assumptions of Cleveland and Liptzin (2007), who suggested that C/PMB ratios in forest floor horizons would be generally higher than those of the underlying mineral soil, due to higher SOC/total P ratios in forest floors. However, soil and site specific characteristics may explain the highest C/PMB ratios, which were measured at the Meissner and the Oerrel site. The Oerrel site is a P depleted sandy Podzol formerly vegetated with heath. C/PMB

173

ratios of up to 50 have been recorded for heathland soils (Nielsen et al., 2009), probably caused by low P availability from Calluna derived SOC (van Meeteren et al., 2007), which is still present in the mineral soil. The strong presence of Fe and Al oxides is most likely the reason for the high C/PMB ratios at the Meissner site, a basalt derived Andosol (Khan and Joergensen, 2012). High contents of sesquioxides may reduce P availability not only by sorption of orthophosphate but also by stabilization of organic P compounds (Celi et al., 2001; Guppy et al., 2005; Yan et al., 2014). In agreement with these considerations, Frossard et al. (2016) suggested that the microbial C/N/P stoichiometry in mineral soils is affected by soil properties rather than by the stoichiometry of the inputs. 5. Conclusions Fumigation extraction was highly suitable for measuring CMB, NMB, and PMB in forest floor horizons after slight modifications, i.e. the ratio of extractant to DM must be sufficiently increased. In this case, Bray-I was a useful extractant for PMB in all the current soil samples, despite the difference in mineralogy. CMB and PMB contents were higher in forest floors under beech than under spruce, but there were no tree species effects on C/PMB ratios. Mean stocks of PMB and total P were roughly 27 and 100 kg ha1 in the forest floor, respectively, but did not differ between the tree species, due to an increased organic matter accumulation in the forest floor under spruce. PMB was closely related to total P, in contrast, but more affected by C availability and CMB in F and H horizons. No evidence was found that PMB contents in mineral A horizons were controlled by C and P availability. The C/PMB ratios declined with increasing decomposition from L to H horizons, but increased again in most A horizons. This was presumably due to strong effects of soil properties, such as pH or presence of Al and Fe oxides. The range of C/PMB ratios was small at all sites in relation to the wide SOC/total P ratios of the litter used as microbial substrate, indicating a relatively strict homeostatic regulation of the forest floor microbial, mainly fungal biomass stoichiometry. Acknowledgements We thank Gabriele Dormann and Anja Sawallisch for their technical advice and assistance. Ulrike Talkner was supported by a DFG grant (TA 826/2-1) associated to the DFG priority program “SPP 1685 e Ecosystem Nutrition: Forest Strategies for Limited Phosphorus Resources”. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2017.04.009. References Achat, D.L., Bakker, M.R., Morel, C., 2009. Process-based assessment of phosphorus availability in a low phosphorus sorbing forest soil using isotopic dilution methods. Soil Science Society of America Journal 73, 2131e2142. Achat, D.L., Augusto, L., Bakker, M.R., Gallet-Budynek, A., Morel, C., 2012. Microbial processes controlling P availability in forest spodosols as affected by soil depth and soil properties. Soil Biology & Biochemistry 44, 39e48. Achat, D.L., Bakker, M.R., Saur, E., Pellerin, S., Augusto, L., Morel, C., 2010a. Quantifying gross mineralisation of P in dead soil organic matter: testing an isotopic dilution method. Geoderma 158, 163e172. , S., Morel, C., 2010b. LongAchat, D.L., Bakker, M.R., Zeller, B., Pellerin, S., Bienaime term organic phosphorus mineralization in Spodosols under forests and its relation to carbon and nitrogen mineralization. Soil Biology & Biochemistry 42, 1479e1490. Achat, D.L., Morel, C., Bakker, M.R., Augusto, L., Pellerin, S., Gallet-Budynek, A., Gonzalez, M., 2010c. Assessing turnover of microbial biomass phosphorus: combination of an isotopic dilution method with a mass balance model. Soil Biology & Biochemistry 42, 2231e2240.

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