Soil Chemical and Microbial Properties in a Mixed Stand of Spruce and Birch in the Ore Mountains (germany)—a Case Study

A major argument for incorporating deciduous tree species in coniferous forest stands is their role in the amelioration and stabilisation of biogeochemical cycles. Current forest management strategies in central Europe aim to increase the area of mixed stands. In order to formulate statements about the ecological effects of mixtures, studies at the stand level are necessary. In a mixed stand of Norway spruce (Picea abies (L.) KARST.) and silver birch (Betula pendula ROTH) in the Ore Mountains (Saxony, Germany), the effects of these two tree species on chemical and microbial parameters in the topsoil were studied at one site in the form of a case study. Samples were taken from the O layer and A horizon in areas of the stand influenced by either birch, spruce or a mixture of birch and spruce. The microbial biomass, basal respiration, metabolic quotient, pH-value and the C and N contents and stocks were analysed in the horizons Of, Oh and A. Significantly higher contents of microbial N were observed in the Of and Oh horizons in the birch and in the spruce-birch strata than in the stratum containing only spruce. The same was found with respect to pH-values in the Of horizon and basal respiration in the Oh horizon. Compared to the spruce stratum, in the birch and spruce-birch strata, significantly lower values were found 1950 for the contents of organic C and total N in the A horizon. The findings of the case study indicated that single birch trees have significant effects on the chemical and microbial topsoil properties in spruce-dominated stands. Therefore, the admixture of birch in spruce stands may distinctly affect nutrient cycling and may also be relevant for soil carbon sequestration. Further studies of these functional aspects are recommended.


Introduction
In central Europe, pure coniferous stands are today viewed very critically, whereas mixed stands are recommended for a variety of reasons.The establishment of mixed stands of Norway spruce (Picea abies (L.) KARST.) and naturally regenerated silver birch (Betula pendula ROTH) and downy birch (Betula pubescens EHRH.) is a common alternative to pure stands of conifers in northern Europe and Asia (e.g., Finland, Norway, Sweden, Russia), but also in central Europe (e.g., Germany, Poland, Czech Republic).Reasons for the growing interest in such stands are, for example, a shift to a greater above-ground nutrient content associated with species grown in mixtures compared to monocultures, indicating an increase in the proportion of resources captured from a site [1], discussions about biodiversity, such as understorey vegetation diversity (e.g., [2]), sequestration of carbon against the background of climate change (e.g., [3]) and forest growth and yield (e.g., [4]).The growth and vitality of trees are dependent on soil conditions, just as soil development is affected by tree species and forest stand type (e.g., [5][6][7][8]).Research has been conducted to examine topsoil properties in pure stands of silver birch and Norway spruce [9][10][11][12][13] and the impact of mixed leaf litter on topsoil has also been studied (e.g., [10,14]).On sites with silver birch and Norway spruce, higher pH values are frequently observed under birch (e.g., [10][11][12]15]).Several studies also identified lower C/N ratios (indicating faster litter decomposition and soil organic matter turnover) in mixed spruce-birch stands than in pure spruce forests (e.g., [12]).In contrast, Hagen-Thorn et al. [13] detected no significant differences between the C/N ratios in the mineral topsoils of 30-40 year old birch and spruce stands.The approaches, conditions and results of different studies are very diverse.The diversity in the results may be related to site conditions (e.g., texture, soil type and climate), stand ages, prior land-use or to the methods employed.
Studies with a focus on soil biological activity (e.g., [16][17][18]) provided important information on the impact of birch in spruce stands.It appears that birch has a positive influence on microbial biomass and on the mass of earthworms, and also affects the structure of microbial communities [10,17,19], for example, through the influence of birch leaves [10].The importance of stand age should also be noted in this context, not only as it influences tree dimensions, but also in relation to the duration of the influence of the trees on topsoil properties (e.g., [16]).Examining biological activity, Miles [16] detected higher numbers of earthworms per m 2 in medium aged and older stands of Betula pendula in Scotland than in young stands.Saetre et al. [17] found higher densities of Coleoptera, Diptera and Colembola in mixed stands than in spruce stands.This is important as soil biota play a key role in the decomposition of litter (e.g., [20][21][22]).Animal communities are also strongly affected by tree species (e.g., [23]).Priha et al. [18] presented data from studies of birch and spruce plots where the content of microbial carbon (Cmic) revealed by fumigation extraction was generally higher under birch than under spruce, but these differences were not significant.At present, it is not possible to draw conclusions on how a mixture of birch and spruce should be designed in order to exert certain effects on the topsoil properties.To address this important question, it appears reasonable to first analyse the effects of single trees.Through the analysis of the effects on topsoil properties of single birch trees in spruce-dominated stands carried out as part of this study, we sought to test the following hypothesis:  Even single birch trees in spruce stands can modify topsoil properties.

Study Site
The study object was a mixed stand of Norway spruce and silver birch on "old" forest soil in the Ore Mountains near Olbernhau/Ansprung, Saxony, Germany (50°38′ N, 13°17′ E).The investigated stand was situated at 725 to 740 m asl with a slight northerly exposition (<5%).The mean annual rainfall was 977 mm and the mean annual temperature 5.8 °C (period 2002-2008; source: Staatsbetrieb Sachsenforst).The stand consisted of Norway spruce with an age of approx.60-80 years and was supplemented to the south by individual silver birch trees, as well as small and large group mixtures with an age of ~60 years (Table 1).The spruce was planted with the aim of establishing a pure stand.Birch regenerated naturally in gaps where spruce planting failed.In areas with more birch trees, the soil had a higher stone content and was not comparable to that of the plots with spruce.For the purposes of the analysis, only single birches situated where the soil properties were comparable to those of spruce plots were selected.The study stand was surrounded by spruce stands.Some isolated beech trees stood in the neighbouring stands, but these were situated far from the chosen study points and could not influence the results.In the neighbouring stands, typical admixed species were birches and mountain-ash (Sorbus aucuparia L.).In densely stocked areas of the study stand, ground vegetation was absent or only poorly developed.Even in sparsely stocked, unshaded areas the ground vegetation was not lush or densely developed.The dominant species were Calamagrostis villosa and Deschampsia flexuosa.
The soil was typical of many mid-range mountains in central Europe: a Dystric Cambisol formed from periglacial debris layers consisting of the weathered products of a gneiss bedrock.The soil chemistry was characterised by high acidity, a low cation exchange capacity and base saturation.The stone content was very high (Table 2).CECeff=effective cation exchange capacity; Corg=organic Carbon; L = Litter; Of = Organic layer with fermentation; Oh = humified organic layer = strongly decomposed organic matter; Ahe = uppermost horizon of the mineral soil enriched with soil organic matter and characterized by eluviation (bleached by podzolization); Ae = mineral horizon of the topsoil characterized by eluviation; Bsh/Bhs = mineral subsoil horizon with Fe/Al-enrichment (sesquioxides) by podzolization and with humus accumulation; Bv = mineral subsoil horizon, weathered, brown and loamy; Cv = weakly weathered parent material

Experimental Design
The approach adopted aimed at investigating the ecological impact of incorporating birch trees into spruce stands, by identifying areas influenced mostly by birch, both by birch and spruce and mostly by spruce trees.Thirty sample plots were assigned to the different strata, according to the species occupying the canopy overhead.All sample points that lay directly beneath the crown of a birch were assigned to the birch stratum (n = 7).Points under the crowns of spruce trees were assigned to the spruce stratum (n = 13).All points located between the crown of a birch and a spruce, and where crowns overlapped, were assigned to the spruce-birch stratum (n = 10).This stratification was used because crowns directly influence the environmental variables (e.g., precipitation throughfall), which in turn affect topsoil properties (e.g., [24,25]).The ecological effects could be measured in terms of their quality and intensity.Usually a gradient in the intensity, and also the quality, of the effects coinciding with the distance from the birch tree can be expected.In stands with more than one tree species, the overlapping zones of the different tree species' effects are of particular interest.In these zones, different litter mixtures exist.This means that two tree species influence the topsoil conditions to different degrees of intensity.Three different tree-to-tree-transects were laid out for this study.All of the transects originated at the same birch tree (Figure 1) and pointed in different directions, representing various tree species mixture gradients.A total of 30 sampling points were then clustered according to whether they were mostly influenced by either birch or spruce trees, or by a mixture of both species.Four different birch trees influenced the sampling sites to varying degrees.The size of the study area within the chosen stand was circa 0.4 ha.
This study was designed as a case study so that further investigations are needed for generalisation of the results beyond the study site.The focus of this study was on an intra-stand level approach for the following reasons: The intra-stand level was deemed to be an appropriate spatial scale at which to optimise the forest structure in plantation forests in order to address new challenges as timber production, neighbour competition and nutrient cycle processes all take place at this scale [26].Single trees are key structural elements of a stand as species, dimension, morphology and the spatial distribution of single trees influence the biotic and abiotic conditions of the intra-stand environment, in particular the understorey vegetation and the topsoil properties (e.g., [27][28][29][30][31][32]).

Soil Sampling and Analysis
To ensure relatively homogeneous soil conditions along the transects, sample points (30 cm × 30 cm) were selected where there were no disturbances (e.g., lying trunks, stumps).Therefore, the distance between the points along the transects was variable (mean distance within one transect = 2.03 m, minimum distance = 0.73 m, maximum distance = 11.5 m).In early September 2008, 90 soil samples were taken from the forest floor (30 samples of Of and 30 samples of Oh horizons) and mineral topsoil (30 samples of A horizon) for chemical analysis in the laboratory.
Fresh soil samples were sieved with a 5 mm sieve for removal of components such as branches, roots and stones.The soil pH was measured in water (50 mL demineralised pure H2O and 10 mL dried soil (dried at 40 °C)) with a glass electrode (Multilab 540 WTW GmbH).The total C and N contents of ground soil samples were measured using the CNS elemental analyser (Vario EL).As the soil had been limed in the past, the total carbon (Ctot) may not have equalled the organic C (Corg ).Therefore, Ccarbonate was also analysed using the gas-volumetric method according to Scheibler (Deutsches Institut für Normung e. V. (DIN) International Organization for Standardization (ISO) 10693).Organic C was calculated as the difference between Ctot and Ccarbonate.The C/N ratio was calculated by dividing Corg by Ntot.Carbon and nitrogen in the microbial biomass (Cmic and Nmic) were analysed using chloroform-fumigation-extraction according to Vance et al. [33] and Joergensen [34].The samples were sieved and analysed fresh.They were not frozen, but cooled to around 4 °C for a maximum of 18 days before the analyses began.After fumigation, shaking with K2SO4 solution for 30 minutes with 180 rotations min −1 and filtration, the C and N contents were determined with the Multi-NC-Analyser (Analytik Jena).To represent the soluble part of C and N, C and N were also extracted from unfumigated soil [33].In accordance with Jenkinson et al. [35], a correction factor of 0.45 was used in the denominator when calculating Cmic and Nmic.The basal respiration was determined using the method described by Isermeyer [36] and modified in accordance with Jäggi [37].To describe the soil profile, the effective cation exchange capacity (CECeff) was analysed based on the NH4Cl extraction method developed by Trüby and Aldinger [38].To calculate the stocks of Corg, Ntot, Cmic and Nmic, the soil bulk densities were determined based on undisturbed soil cores taken using soil sample rings (for the O layer: sampling rings with a diameter of 7.2 cm and a volume of 248.4 cm 2 , for the mineral sampling rings with a diameter of 5.5 cm and a volume of 95 cm 2 ).The samples were dried at 60 °C (forest floor) and 105 °C (mineral soil) for 24 h.The thickness of the sampled soil horizons was the average of three measurements around the soil cores.The mean thicknesses of the Of and Oh horizons in the three different strata were: birch: Of = 0.97 cm, Oh = 1.83 cm, spruce-birch: Of = 1.23 cm, Oh = 2.47 cm and spruce: Of = 1.30cm, Oh = 2.40 cm.Coarse material was removed using a 2 mm mesh and the mass fraction of the horizon area (g m −2 ) was determined.

Statistical Analysis
An extended linear model without random effects was applied to the data.The model validation process outlined by Zuur et al. ([39]; p19-p21) was followed and the normality, the homogeneity and the independence in the dataset assessed.To be certain of normality, all residuals of the applied model were pooled and their distribution inspected (by q-q-plots).Heteroscedasticity was assessed by pooling all residuals and plotting them against the fitted values.Where heteroscedasticity was an issue, the model was adapted to account for the variance structure of the data.The spatial independence of the data was assessed employing the semivariogram technique ( [40]; p. 226).Where necessary, a spatial dependence structure was incorporated in the model.P-values were adjusted in simultaneous multiple testing by applying the Benjamini-Hochberg procedure ( [41]; p. 534).As natural heterogeneity is high in forest soils, and the sample size low, an error probability of α = 0.10 was applied and the significant differences observed are reported and discussed as trends.An error probability of α = 0.10 is frequently used for studies of microbial properties in soils (e.g., [42][43][44]).All statistical analyses were performed using R and boxplots were created with SPSS.

Carbon
Significant differences in the Corg content occurred between the tree strata in the A horizon (Figure 2a).Here birch < spruce (p = 0.066; medians: 71 mg g −1 < 118 mg g −1 ) and spruce-birch < spruce with (p = 0.066; medians: 103 mg g −1 < 118 mg g −1 ).A decrease in the Corg content was observed from the Of horizon to the A horizon.
The Corg stocks (Figure 2b) did not differ between the three strata within each horizon analysed.The highest values were found in the Oh horizon and the lowest in the A horizon.

Nitrogen
No significant differences existed for the Ntot contents in the Of and Oh horizons (Figure 2c).Ntot contents were lowest in the A horizon, where they differed significantly between the three strata, with birch < spruce (p = 0.087; medians: 4.24 mg g −1 < 6.06 mg g −1 ) as well as spruce-birch < spruce (p = 0.087; medians: 5.45 mg g −1 < 6.06 mg g −1 ).
There were no significant differences in the Of, Oh or A horizons between the strata for the Ntot stocks (Figure 2d).The Ntot stocks were higher in the Oh horizon than in the Of and the A horizons.

C/N Ratio and Acidity
No noticeable differences were found for the C/N ratio (Figure 2e).It was notable that the C/N ratios in the Oh horizon were lower than in either the Of horizon or the A horizon.
The Of horizon exhibited a significantly higher pH-value (Figure 2f) in the birch stratum (median = 5.41) than in the spruce (median = 5.12) with p = 0.086, and in the spruce-birch stratum (median = 5.49) than in the spruce with p = 0.086.No noticeable differences were found in the Oh and A horizons.The highest pH-values were found in the Oh horizon, the lowest in the A horizon.

Microbial Properties
The results of the analysis of Cmic contents (Figure 3a) revealed no notable differences in all three horizons.Cmic contents decreased from the Of horizon to the A horizon.
The analysis of the metabolic quotient (qCO2), an indicator of the CO2 release rate per unit of microbial biomass ((mg CO2-C h −1 ) g −1 Cmic), revealed no significant differences (Figure 3d).The metabolic quotient was highest in the Of horizon, which also had the greatest variation.There the spruce stratum exhibited the second lowest and the birch stratum the lowest values.

Discussion
Tree species can affect soil properties in different ways; for example, prompting differences in the chemical composition of above-and below-ground litter, differences in root activity and changes in microclimate under the tree cover, changing the understorey vegetation [45].The aim of this study was to assess whether even single tree admixtures of birch have a distinct effect on topsoil properties in spruce stands.The results of the stratified analysis provided evidence that when silver birch and Norway spruce grew in the same stand, the pH-value in the Of horizon was less than a half unit higher under birch.These findings were consistent with previous studies (e.g., [25,46,47]).Augusto et al. [48] determined that the pH-value of topsoil from the same site is on average about 0.43 units higher under birch than under spruce.A higher pH of this magnitude could positively affect the microbial biomass in the soil (see below) and the number and mass of earthworms [49].Such changes in pH may also influence the roots of spruce.Both the growth and the morphology of spruce roots depend greatly on soil chemical properties affecting the composition of soil solution [50].Acid conditions inhibit root growth (e.g., [51]) and lead to fewer fine roots in the mineral soil, and to a concentration of the roots in the O layer (e.g., [52]).The presence of the roots of the two different tree species also affect the soil; for example, through root exudates [53].Sandnes et al. [53] concluded that the rhizosphere of birch contains more organic acids at higher concentrations than spruce.Higher organic acid production means a higher potential to lower pH, which contradicts our earlier discussion.Due to the differences in the root systems and root distribution of spruce [54,55] and birch [56], different effects on the soil are likely.The C/N ratio is an important indicator of the transformation of soil organic matter.The observation that C/N ratios were generally lower in the birch and spruce-birch strata than in the spruce stratum corresponded to the results of several earlier studies (e.g., [10,12,[57][58][59]). Material with a lower C/N ratio can be decomposed by microorganisms much more readily.However, in this study the differences between the strata were not significant.
The Ntot contents of the Of and Oh horizons exhibited no significant differences, whereas in the A horizon the values were significantly higher for the spruce stratum than for the birch and spruce-birch strata.Priha and Smolander [60] also found lower Ntot contents under a birch stand than a neighbouring even-aged spruce stand.However, the findings of our study deviated from those of certain other studies such as Lettl and Hysek [9] and Brandtberg et al. [11].Lettl and Hysek [9] found higher Ntot contents in the Of horizon under spruce but lower contents of Ntot in the Oh when compared with birch.Brandtberg et al. [11] found no differences between the two species with respect to Ntot contents in a 20-30 year old spruce-birch stand.This may have been due to the low stand age in their study.Many other studies have produced evidence of a change in soil properties as a function of stand age (e.g., [16,[61][62][63]), time elapsed since the understorey planting of deciduous trees (e.g., [64]) and single tree ages (e.g., [65]).However, in our study, the effects of stand age were not captured at all (stand was even-aged 60-80 years).
The results of our study revealed higher Nmic contents under the influence of birch than under spruce in the O layer.These results were consistent with Priha et al. [18] and Smolander et al. [66].An inverse pattern was found for the A horizon in our study and by Smolander et al. [66].We found greater basal respiration under birch than under spruce in the Oh horizon, similar to Priha et al. [18] and Smolander et al. [66].Our results support the assumption that the presence of birch might increase microbial biomass [10,16,19,67].In our study, no significant difference between the tree species could be found for the three horizons with respect to the metabolic quotient.This was consistent with the findings of Priha and Smolander [68].In the A horizon, Corg contents were lowest in the pure birch stratum and intermediate in the mixed stratum, which corresponded to the lowest C/N ratio, reflecting amongst other things the better birch litter quality compared to spruce litter (e.g., [69]).Johansson [70] proved that at sites where Norway spruce and white birch grew in adjacent stands, the birch leaf litter generally had higher nutrient concentrations.Concerning organic chemical components, the spruce needle litter had significantly higher concentrations of lignin and mannan and lower concentrations of water solubles, rhamnan and xylan than the birch litter [70].Furthermore, Kanerva et al. [47] observed lower concentrations of terpenes under birch in comparison with spruce.In the Oh horizon of our study, the mixed stratum revealed the lowest values of Corg contents (but no significant differences between the strata), indicating a faster turnover of soil organic matter in this horizon.This was not only the effect of litter quality (more favourable for decomposition), as the values in the pure birch stratum were more similar to those of the spruce stratum, but more likely an effect of increased microbial activity, which naturally depends, for example, on litter quality.This may hint at a more diverse and active soil biocoenosis in the Oh horizon in the mixed stratum overall than found in the single-species strata [71].As a consequence, more organic material is incorporated into the mineral soil in the mixed stratum, which is reflected in the highest Corg stocks in the A horizon of the mixed stratum, although the differences between the strata were not significant.However, some of our results indicate that even single tree admixtures of birch have a distinct effect on topsoil properties in spruce stands.Therefore, we accept the hypothesis that even single birch trees in spruce stands can modify topsoil properties.It must be stated in relation to this, however, that our results could have been influenced by factors such as the anisotropy in the distribution of birch litter [32] caused by the main wind direction (south-west, Figure 1), the small-scale variability of throughfall and corresponding differences with respect to water supply (e.g., [72]), the fine root dynamics (e.g., [73]) or the diversity of radiation in the stand and the related differences in soil temperature and light availability for the ground vegetation [32].These factors influence the variance of data and might lead to less significant differences between the strata.

Conclusions
The findings of this study show that even single tree admixtures of birch have a distinct effect on topsoil properties in spruce stands.The admixture of single birch trees generates higher pH-values and higher microbial activity in the O layer.The admixture of single birch trees in spruce stands may also play a role in soil carbon sequestration, because higher microbial activity could lead to lower total soil organic carbon (SOC) stocks in the humus layer but higher and more stable SOC stocks in the mineral soil.Future research should investigate whether the higher biological activity in soils causes greater incorporation of humus into the upper mineral soil and study the pools of stable and unstable organic carbon in spruce-birch mixtures in order to analyse the real potential for carbon sequestration.One might expect that an increase in the number of birch trees leads to an intensification of the observed effects.Another important aspect for future research might be a study of whether different ages of litter affect the different topsoil results (e.g., soil microbial properties) in such mixtures.That is a very interesting question because partly decomposed spruce litter of different ages could be found in the Of horizon (typical for conifers), while birch litter consisted mainly of the leaves from the last autumn (typical for many broadleaves), indicating a different state of decay and thus possibly a different composition of the litter remains.As our findings indicated that single birch trees have significant effects on the chemical and microbial topsoil properties in spruce-dominated stands, the admixture of birch in spruce stands may have a distinct effect on nutrient cycling and may also be relevant for soil carbon sequestration.Consequently, further studies of these functional aspects are recommended.

Figure 1 .
Figure 1.Map of tree stems and crowns with soil sampling points indicated along transects with percental distribution of the wind direction (01.01.1997-31.05.2009) measured at the Olbernhau permanent monitoring area (Staatsbetrieb Sachsenforst); average wind speed: 1.51 m s −1 , maximum wind speed: 6.41 m s −1 .

Table 1 .
Description of the spruce-birch stand studied in the Ore Mountains, Germany.

Table 2 .
Description of the soil physical and chemical properties for the study site (samples came from an extra soil profile created for the characterisation of the study site), n.a.= not analysed.