The linkage of 13C and 15N soil depth gradients with C:N and O:C stoichiometry reveals tree species effects on organic matter turnover in soil

Abstract

The knowledge of tree species dependent turnover of soil organic matter (SOM) is limited, yet required to understand the carbon sequestration function of forest soil. We combined investigations of 13C and 15N and its relationship to elemental stoichiometry along soil depth gradients in 35-year old monocultural stands of Douglas fir (Pseudotsuga menziesii), black pine (Pinus nigra), European beech (Fagus sylvatica) and red oak (Quercus rubra) growing on a uniform post-mining soil. We investigated the natural abundance of 13C and 15N and the carbon:nitrogen (C:N) and oxygen:carbon (O:C) stoichiometry of litterfall and fine roots as well as SOM in the forest floor and mineral soil. Tree species had a significant effect on SOM d13C and d15N reflecting significantly different signatures of litterfall and root inputs. Throughout the soil profile, d13C and d15N were significantly related to the C:N and O:C ratio which indicates that isotope enrichment with soil depth is linked to the turnover of organic matter (OM). Significantly higher turnover of OM in soils under deciduous tree species depended to 46% on the quality of litterfall and root inputs (N content, C:N, O:C ratio), and the initial isotopic signatures of litterfall. Hence, SOM composition and turnover also depends on additional-presumably microbial driven-factors. The enrichment of 15N with soil depth was generally linked to 13C. In soils under pine, however, with limited N and C availability, the enrichment of 15N was decoupled from 13C. This suggests that transformation pathways depend on litter quality of tree species.

Full text

The linkage of
13
C and
15
N soil depth gradients with C:N
and O:C stoichiometry reveals tree species effects on organic
matter turnover in soil
Marcel Lorenz .Delphine Derrien .Bernd Zeller .Thomas Udelhoven .
Willy Werner .So
¨ren Thiele-Bruhn
Received: 18 May 2020 / Accepted: 30 October 2020 / Published online: 12 November 2020
ÓThe Author(s) 2020
Abstract The knowledge of tree species dependent
turnover of soil organic matter (SOM) is limited, yet
required to understand the carbon sequestration func-
tion of forest soil. We combined investigations of
13
C
and
15
N and its relationship to elemental stoichiometry
along soil depth gradients in 35-year old monocultural
stands of Douglas fir (Pseudotsuga menziesii), black
pine (Pinus nigra), European beech (Fagus sylvatica)
and red oak (Quercus rubra) growing on a uniform
post-mining soil. We investigated the natural
abundance of
13
C and
15
N and the carbon:nitrogen
(C:N) and oxygen:carbon (O:C) stoichiometry of
litterfall and fine roots as well as SOM in the forest
floor and mineral soil. Tree species had a significant
effect on SOM d
13
C and d
15
N reflecting significantly
different signatures of litterfall and root inputs.
Throughout the soil profile, d
13
C and d
15
N were
significantly related to the C:N and O:C ratio which
indicates that isotope enrichment with soil depth is
linked to the turnover of organic matter (OM).
Significantly higher turnover of OM in soils under
deciduous tree species depended to 46% on the quality
of litterfall and root inputs (N content, C:N, O:C ratio),
and the initial isotopic signatures of litterfall. Hence,
SOM composition and turnover also depends on
additional—presumably microbial driven—factors.
The enrichment of
15
N with soil depth was generally
linked to
13
C. In soils under pine, however, with
limited N and C availability, the enrichment of
15
N
was decoupled from
13
C. This suggests that transfor-
mation pathways depend on litter quality of tree
species.
Keywords Stable isotopes Microbial turnover 
Litter Roots Common garden experiment 
Recultivated forest soil
Responsible Editor: Edith Bai.
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10533-020-00721-3) con-
tains supplementary material, which is available to authorized
users.
M. Lorenz S. Thiele-Bruhn (&)
Soil Science Department, FB VI, University of Trier,
Behringstrasse 21, 54296 Trier, Germany
e-mail: thiele@uni-trier.de
D. Derrien B. Zeller
INRAe Grand Est Nancy, UR 1138 Bioge
´ochimie des
E
´cosyste
`mes Forestiers, 54000 Nancy, France
T. Udelhoven
Environmental Remote Sensing & Geoinformatics
Department, FB VI, University of Trier, Behringstrasse
21, 54296 Trier, Germany
W. Werner
Geobotany Department, FB VI, University of Trier,
Behringstrasse 21, 54296 Trier, Germany
123
Biogeochemistry (2020) 151:203–220
https://doi.org/10.1007/s10533-020-00721-3(0123456789().,-volV)(0123456789().,-volV)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Introduction
Soils in forest ecosystems bear a high potential as
carbon (C) sinks in the mitigation of climate change
(Pan et al. 2011). Tree species identity plays a crucial
role in the C cycle of these ecosystems, e.g. by fueling
soil with bio- and necromass, respectively (Augusto
et al. 2015). The total amount of C that is stored in the
forest floor and mineral soil is affected by the
dominating tree species (Mueller et al. 2015). Fur-
thermore, stoichiometric ratios like the carbon:nitro-
gen (C:N) ratio of soil organic matter (SOM) are
influenced by the forest stand (Cools et al. 2014;
Lorenz and Thiele-Bruhn 2019). Ecological stoi-
chiometry using elemental ratios is a suitable tool to
assess SOM and its turnover (Manzoni et al. 2010;
Zechmeister-Boltenstern et al. 2015). For this, the C:N
ratio is commonly used (Stevenson 1994). Also the
oxygen:carbon (O:C) ratio bears high potential to
characterize SOM because it reflects the state of
oxidation of SOM (Beyer et al. 1998). Furthermore,
the abundance of stable isotopes (
13
C and
15
N) in soils
also provide a powerful tool for investigating spatial
and temporal SOM dynamics (Ehleringer et al. 2000;
Bru
¨ggemann et al. 2011; Craine et al. 2015). Varia-
tions in the isotopic composition are useful for tracing
carbon sources and fluxes between plants, microor-
ganisms and soils, thus serving to elucidate the impact
of plant inputs on SOM formation (Balesdent et al.
1987). A combination of both approaches, ecological
stoichiometry and stable isotopes, in soil depth
gradients promises to get deeper insights into the
turnover of SOM. To our knowledge, this has not been
used before to characterize the turnover of tree species
dependent organic matter (OM) in the soil.
Typically,
13
C and
15
N show trends of enrichment
with increasing soil depth that were related to aging
and turnover of OM (Nadelhoffer and Fry 1988;
Billings and Richter 2006; Trumbore 2009). Several
SOM turnover and stabilization mechanisms were
identified that can lead to a variation of the natural
abundance of
13
C. Litter with lower d
13
C values from
aboveground plant materials triggers the topsoil, while
the contribution of
13
C-enriched root inputs to SOM
d
13
C increases with increasing soil depth (Bird et al.
2003). Root inputs encompass C release from plant
roots to soil including: (1) root cap and border cell
loss, (2) necromass from root cells and tissues, (3) C
flow to root-associated, soil living symbionts (e.g.
mycorrhiza), (4) gaseous losses, (5) root exudates, and
(6) mucilage (Jones et al. 2009). During the microbial
metabolism of C, preferentially
13
C-depleted mole-
cules will be respired by microorganisms and the
remaining SOM will be
13
C-enriched (Lerch et al.
2011). In general, microorganisms are
13
C-enriched
compared to plant material or bulk SOM (Dijkstra
et al. 2006) and the contribution of microbial derived
C increases with the extent of OM turnover (Bostro
¨m
et al. 2007). Additionally, OM associated with soil
minerals is characterized by increased d
13
C values
compared to free or occluded light OM fractions
(Schrumpf et al. 2013). The association of OM with
minerals is an important mechanism for its stabiliza-
tion in soil (von Lu
¨tzow et al. 2007). The prevalence of
SOC decrease and d
13
C increase with depth in well-
drained forest soils has prompted the use of the
gradient of SOC plotted against d
13
C as a proxy for
SOM turnover (Acton et al. 2013). Consequently,
depth-related interconnection of d
13
C and SOC
describes the rate of change in
13
C natural abundance
along a decay continuum from fresh litter inputs to
more decomposed SOM (Garten et al. 2000).
The absolute enrichment of
15
N over soil depth can
be determined as the difference between the maximum
enrichment of
15
N in the mineral soil and the litter
bearing OL horizon (Hobbie and Ouimette 2009). The
development of
15
N with soil depth is related to N
cycling processes that are coupled to SOM turnover
(Emmett et al. 1998). Similar to d
13
C values, organo-
mineral associations (Kramer et al. 2017) and the
accumulation of
15
N enriched microbial biomass in
more transformed SOM (Wallander et al. 2009) can
drive the d
15
N patterns within soil. Furthermore, the
type and degree of mycorrhizal associations (Hobbie
and Ho
¨gberg 2012), enzymatic hydrolysis (Silfer et al.
1992), N losses after ammonification, nitrification and
denitrification (Ho
¨gberg 1997;Po
¨rtl et al. 2007),
atmospheric depositions (Vallano and Sparks 2013)
and mixing of soil N through bioturbation (Wilske
et al. 2015) contributes to the
15
N enrichments along
the soil profile. Both d
13
C and d
15
N are mechanisti-
cally linked through the decomposition and microbial
processing of SOM (Nel et al. 2018), thus highlighting
the suitability of both parameters to determine the
degree of organic matter turnover in soil.
In natural mixed forest ecosystems it is difficult to
track down a tree species effect on SOM status;
therefore common garden experiments, where
123
204 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

different tree species were planted in adjacent blocks
at the same time on similar soil, were established to
study tree species effects (Reich et al. 2005; Vesterdal
et al. 2013). Important insights into the relationship
between tree species and the cycling of soil C and
other nutrients in forest ecosystems were gained from
common garden experiments (e.g. Mueller et al. 2012;
Gurmesa et al. 2013). Nevertheless, such experiments
are often handicapped by a previous land-use conver-
sion, e.g. from arable land or from clear felled forests
(Vesterdal et al. 2008). Old SOM from former land-
use types often makes the interpretation of the effects
of tested species and their SOM on SOM dynamics
rather difficult (Balesdent et al. 2018). Callesen et al.
(2013) revealed in a common garden experiment that
the patterns of d
15
N in soil profiles reflected the former
arable land-use type. Comparable with common
garden experiments, differently afforested soils at
post-mining sites provide a unique opportunity for
understanding mechanisms in SOM formation (Frouz
et al. 2009). In particular, sites that are free from old C
sources can be suitable but investigations on such sites
are rare. Due to this, further research is required to
clarify if d
13
C and d
15
N, and thus the SOM status in
organic forest floor horizons (litter—OL, frag-
mented—OF, humified—OH) and mineral soil differs
between tree species.
This research was conducted on a post-mining site,
where previous accumulation of plant or coal material
are negligible (Lorenz & Thiele-Bruhn 2019). We
studied monocultural stands of Douglas fir (Pseudot-
suga menziesii), black pine (Pinus nigra), European
beech (Fagus sylvatica) and red oak (Quercus rubra)
that were grown for 35 years under identical soil and
geomorphological conditions to assess tree species
effects on the SOM status. In more detail, we
investigated the natural abundance of
13
C and
15
Nin
combination with C:N and O:C stoichiometry of
litterfall and root inputs (determined as belowground
phytomass) as well as SOM in depth gradients of
forest soils to answer the following questions:
(1) Do litterfall and root inputs differ in their
isotopic signatures of d
13
C and d
15
N between
tree species?
(2) Is there a tree species effect on d
13
C and d
15
Nin
the depth gradients starting from the OL horizon
down to 10–30 cm of mineral soil?
(3) Are stable isotope contents in the depth profiles
related to the stoichiometry (C:N and O:C ratio)
of the bulk soil?
(4) Varies the decomposition of OM and the
stabilization of it in soil significantly between
tree species and if yes, are litterfall and/or root
properties important for these processes?
Materials and methods
Study site
The study was conducted at the afforested spoil heap
‘Sophienho
¨he’, located in the northwest of the lignite
open-cast mine ‘Hambach’ in the Rhineland, Germany
(N 50°56.110,E6°26.560). There, boundary condi-
tions regarding soil, climate, topography and manage-
ment were highly similar, equivalent to a common
garden experiment. The Regosols at the investigated
sites developed on the same sandy gravelly parent
material (Lorenz and Thiele-Bruhn 2019). The car-
bonate-free parent material that was used for the spoil
heap recultivation had a C content of 0.20 ±0.05%
and a C/N molar ratio of 7.5 ±1.2 (Table S1).
Therefore, a relevant impact of old or fossil carbon
from former land use types and the introduction of coal
from lignite mining was excluded (Lorenz and Thiele-
Bruhn 2019). The investigation was carried out in
monocultural stands of Douglas fir (Pseudotsuga
menziesii), black pine (Pinus nigra), European beech
(Fagus sylvatica) and red oak (Quercus rubra) that
were afforested in 1982 on the western slopes of the
spoil heap. Within 35 years after the start of the
afforestation organic layers had developed that were
classified as Moder (Zanella et al. 2018) with slight
differences between tree stands. Dependent on the
thickness of the OH layer, Dysmoder was the dom-
inant humus form that coexisted in some patchy
sections with Eumoder under Douglas fir, beech and
oak, while under pine solely Dysmoder had developed
(Lorenz and Thiele-Bruhn 2019).
Sampling scheme and sample preparation
Each species stand is subdivided in six to ten plots with
a size of 1780 ±660 m
2
by skid trails established in
slope line. For each of the four stands, five plots were
123
Biogeochemistry (2020) 151:203–220 205
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

selected for sampling of the forest floor, mineral soil,
roots and litterfall (Fig. 1). Sampling points (light grey
circles) within one plot were located at least 25 m
away from forest roads and in the upper parts of the
middle slopes. In April 2016, forest floor samples were
taken with a steel frame (20 cm 920 cm) and
carefully separated into the organic litter, fragmented,
and humified horizon, OL, OF and OH, respectively,
according to Zanella et al. (2018). Afterwards, bulk
soil samples were taken at three different depths
(0–5 cm, 5–10 cm, 10–30 cm) from excavated
50 cm 950 cm 950 cm pits. To ensure representa-
tiveness, forest floor and soil samples (grey rectangles)
were taken from four positions and samples from
similar depths were subsequently pooled. In total 60
forest floor samples and 60 mineral soil samples were
collected (five per depth in each stand, Table S2),
transported and stored at 4 °C for further preparation.
Forest floor samples were dried at 60 °C and visible
roots were carefully sorted out. Mineral soil samples
were passed through a 2 mm sieve, roots were
removed and the soil samples were dried at 60 °C.
All samples were ground and homogenized using a
ball mill (Retsch MM400, Retsch GmbH, Haan,
Germany).
We performed root sampling 2 years later, in April
2018 and distinguished roots from different horizons.
To do so, we collected five replicate samples of forest
floor and mineral soil (dark grey circles in Fig. 1) with
a distance of 1 m around a tree within each of the five
plots per species stand. Forest floor roots were
collected using a steel frame (20 cm 920 cm).
Underneath, mineral soil roots were collected using
a root auger with a diameter of 8 cm (Eijkelkamp Soil
& Water, Giesbeck, Netherlands). The cores of
mineral soil were divided into the three subsamples
(0–5 cm, 5–10 cm. 10–30 cm). In total, we collected
400 root samples (25 per depth in each species stand,
Table S2) that were transported and stored at 4 °C. In
the laboratory, the forest floor samples were spread out
in plastic bowls and roots were carefully separated
using a tweezer. The roots were carefully washed to
remove adherent soil particles. The mineral soil
samples were put into plastic bowls and immediately
washed with water to separate roots. Roots with a
diameter B5 mm were dried at 105 °C to determine
dry weights and a subset of 80 samples (five per depth
per species) was homogenized using a ball mill
(Retsch MM400, Retsch GmbH, Haan, Germany) for
further chemical analysis.
In each of the five plots per species stand the
litterfall was collected using litter traps made with
nylon mesh (0.5 mm mesh size) that was fixed on a
wooden frame (1 m 91 m). Litter traps were
Fig. 1 Sampling design at the study site ‘Sophienho
¨he’.
35 year-old afforested monocultural stands of Douglas fir
(Pseudotsuga menziesii), black pine (Pinus nigra), European
beech (Fagus sylvatica) and red oak (Quercus rubra) were
investigated. They are located on the western exposed slopes
(inclination: 22.2°±2.2°) of the spoil heap. Each stand is
subdivided in six to ten plots with a size of 1780 ±660 m
2
by
skid trails established in slope line. For each of the four tree
stands, five plots were selected for sampling (light grey circles)
of the forest floor (grey rectangles), mineral soil (grey
rectangles), roots (dark grey circles) and litterfall (litter trap)
123
206 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

installed 1 m above the soil surface and located in the
plot near the central soil sampling point (Fig. 1). In the
timespan from July 2016 to June 2017 the litter traps
were monthly emptied. In the laboratory, the 240
samples were immediately separated into foliar and
non-foliar fractions and dried at 60 °C to determine
dry weights (Ukonmaanaho et al. 2016). The foliar
fraction of the 12 monthly samples of each litter trap
were pooled into one mixed sample. Consequently,
five litterfall samples per tree species resulted in a total
number of 20 litterfall samples (Table S2) that were
homogenized using a ball mill (Retsch MM400,
Retsch GmbH, Haan, Germany) for further chemical
analysis.
Laboratory analysis
Total contents of C, N and O were determined using an
Elemental Analyser EA3000 (HEKAtech GmbH,
Wegberg, Germany). Soil samples were acidic and
free of carbonate (Lorenz and Thiele-Bruhn 2019),
thus the measured total C content represents organic
C. The contents of the elements were used to calculate
the molar C:N and O:C ratios. The stable isotopes
13
C
and
15
N were determined by an IsoPrime 100 isotope
ratio mass-spectrometer (IsoPrime Corporation, Chea-
dle, UK) and vario ISOTOPE cube elemental analyzer
(Elementar Analysensysteme GmbH, Hanau, Ger-
many). Stable isotope compositions are reported in
delta notation (d
13
C%and d
15
N%) relative to
Vienna Pee-Dee Belemnite (VPDB) for C, using the
international reference materials IAEA-CH-7
(-32.151%VPDB SD ±0.05%) as a standard,
and relative to atmospheric N
2
for N, using IAEA-N-1
(?0.4%air N2 SD ±0.2%), IAEA-N-2 (?20.3%
air N2 SD ±0.2%) and USGS32 (?180%air N2
SD ±1%) as standard according to Eq. (1):
dsample ¼Rsample
Rstandard
1

1000;ð1Þ
where Rrepresents the ratio of
13
C/
12
Cor
15
N/
14
N,
respectively. The measurement error of d
15
N was
approximately 0.2%and \0.1%for d
13
C.
Data processing and statistics
The relationship between the prevalent vertical
decrease of SOC and increase of d
13
C in depth profiles
was used as a natural indicator of SOC turnover
(Acton et al. 2013). The slope (b) of the linear
regression (y=a?bx) between the mean d
13
C
values and their respective log-transformed C con-
centrations (mg C g
-1
) was calculated and is referred
as b
d13C
value. The distribution of
15
N along soil depth
profiles was compared between tree stands using the
soil enrichment factor (e
soil
15
N). It is defined as
absolute enrichment between the OL horizon and the
10–30 cm mineral soil layer (Hobbie and Ouimette
2009) and was calculated following Eq. (2):
esoil15 N&ðÞ¼d15N1030cm d15 NOL:ð2Þ
The following statistical analyses were conducted
separately for litter inputs (litterfall and roots) and
each depth starting from the OL horizon down to
10–30 cm with the R statistical package version 3.3.2.
(R Core Team 2016). Boxplots and one-way analysis
variance (ANOVA) as pretests were carried out to
inspect the data structure. The residuals of ANOVA
were tested for normality and homoscedasticity using
the Shapiro–Wilk test respectively Levene’s test.
Accordingly, normal distributed and homoscedastic
data were tested for significant differences between
tree species by one-way ANOVA followed by the
Tukey’s honest significant difference (HSD) post hoc
test. Significant differences between tree species for
normal distributed but heteroscedastic data were
tested using Welch-ANOVA followed by a pairwise
ttest with Bonferroni–Holm correction. In case data
was not normal distributed but homoscedastic the
Kruskal–Wallis test was applied followed by the Dunn
test. Variance analyses and necessary pretests were
performed with a significance level of p\0.05. The
results are presented in arithmetic mean ±standard
deviation (SD) for the different tree stands. To
characterize relationships between isotopic (d
13
C,
d
15
N,) and stoichiometric (C:N, O:C) parameters
regression analyses with linear and logarithmic func-
tions were done. Additionally, multiple linear regres-
sion models were generated to analyze explaining
variables for b
d13C
and e
soil
15
N values. For this
purpose, C, N, C:N, O:C, d
13
C, d
15
N, as well as the
biomass of litterfall and roots were used as indepen-
dent variables. To simplify the complexity of the
model, parameters were stepwise eliminated that
decrease the quality of the regression model by
assessing R
2
and pvalues. Finally, the most
123
Biogeochemistry (2020) 151:203–220 207
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

appropriate models that comprise only parameters
with significant portions of the explainable variance
were used to discuss driving factors for b
d13C
and
e
soil
15
N values. The results of the regression analyses
were described as significant in cases where p\0.05.
Results
Litter inputs
The total annual foliar litterfall in pine stands was
significantly higher compared to beech stands,
whereas under Douglas fir and oak intermediate
amounts were reached (Table 1). The
13
C content of
litterfall were significantly highest in Douglas fir
stands and declined in the sequence Douglas fir [
pine Coak [beech. Also, d
15
N of litterfall was
significantly higher in Douglas fir stands compared
to the other tree species. The litterfall of coniferous
species was characterized by significantly higher C:N
ratios compared to deciduous forest stands. The O:C
ratio of litterfall decreased in the order
oak [beech [pine [Douglas fir.
In contrast to the litterfall, substantially and in part
significantly higher root biomasses were detected in
the upper 30 cm of soil under deciduous tree species
than under conifers (Table 1). In general, the roots of
all tree species were significantly enriched in
13
Cby
4.56 ±0.99%and
15
N by 3.01 ±0.61%compared
to the litterfall. Douglas fir roots were characterized by
significantly higher d
13
C values compared to those of
pine and beech, while the significantly lowest d
13
C
values were determined in oak roots. Similar differ-
ences between tree stands were found for d
15
N of roots
as well as for litterfall d
15
N. Obviously, the isotopic
signatures of roots in the forest floors did not differ
from roots in deeper soil horizons but the tree species
effect was similarly pronounced in each soil depth. In
contrast to the litterfall, the C:N ratio of beech and oak
roots was significantly higher compared to the conif-
erous species (Table S3). The O:C ratio of roots was
highest in the oak stand similar to the litterfall.
Depth profiles of bulk soil d
13
C and d
15
N and their
relationship to stoichiometry patterns
The parent material for soil recultivation was charac-
terized by a d
13
C value of -29.69 ±0.13%and a
d
15
N value of -0.89 ±0.09%(Table S1). In
general, with increasing soil depth an enrichment of
13
C and
15
N was observed, while the contents of C and
N decreased (Fig. 2). A small deviation from this
pattern occurred for
13
C in the forest floor horizons,
where a depletion or no significant variation from OL
to OH horizon was detected. d
13
C varied in a range
from -29.37%(oak, OF) to -26.19%(Douglas fir,
10–30 cm). The coniferous species Douglas fir and
pine caused significantly higher d
13
C values in the
forest floor compared to beech and oak. In the mineral
soil lowest d
13
C values were found in the oak stands
(-28.13%to -27.32%), while in the Douglas fir
stands highest d
13
C values from -26.91%to
-26.19%were measured (Table S4). Throughout
the soil profile, a significant effect of tree species on
d
13
C was detected (Fig. 2b).
Compared to d
13
C, d
15
N varied in a wider range
from -6.93%(pine, OL) to 0.54%(beech,
10–30 cm) and depth gradients were more pro-
nounced. The forest floor horizons (OL, OF, OH) of
Douglas fir showed significantly higher d
15
N values
compared to the other tree species (Fig. 2d). In the first
two mineral soil layers (0–5 cm, 5–10 cm) the d
15
N
values of all tree species converged, while at a depth
from 10 to 30 cm under beech and Douglas fir
significantly higher values were determined compared
to oak. Consequently, a tree species effect on d
15
N was
found in the forest floor as well as the deepest
investigated mineral soil layer from 10 to 30 cm.
Regression analyses revealed that d
13
C and d
15
N
were related to the C:N and O:C ratio (Table 2). The
relationships of these two stoichiometric ratios of the
bulk soil were stronger with d
15
N than with d
13
C and
were better described by a logarithmic equation rather
than by a linear equation (Tables 2and S5). Along the
soil profile from OL to 10–30 cm depth the C:N ratio
decreased in a range from 52.8 (oak, OL) to 15.7
(beech, 10–30 cm), while the O:C ratio increased from
0.40 (Douglas fir, pine, OL) to 3.07 (beech, 10–30 cm)
(Table S4). With increasing soil depth the decline of
C:N was exponentially correlated to an enrichment of
15
N (Fig. 3a). Different slopes of the regression lines
showed that the relationship between C:N and d
15
N
was differently pronounced dependent on the tree
species. Similarly close regressions were determined
for the relationship between O:C and d
15
N (Fig. 3b).
Yet, curves exponentially increased, showing
15
N
enrichment with increasing O:C ratio.
123
208 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Table 1 Biomass and isotopic composition of litterfall and root inputs of different tree species, 35 years after afforestation on a post-mining site
Roots in
Litterfall Roots (general) Forest floor 0–5 cm 5–10 cm 10–30 cm
Biomass Douglas fir 3.59 ±0.99ab 9.01 ±1.83ab 0.37 ±0.19bc 4.23 ±1.72a 1.38 ±0.54a 3.04 ±0.66
[t ha
-1
] Pine 4.54 ±0.73b 6.73 ±1.89a 0.09 ±0.05a 1.30 ±0.44a 1.68 ±1.02a 3.66 ±1.59
Beech 3.30 ±0.34a 18.41 ±6.88bc 0.61 ±0.10c 4.75 ±2.40a 4.29 ±2.77b 8.76 ±6.51
Oak 3.43 ±0.38ab 22.70 ±7.22c 0.29 ±0.18ab 10.66 ±3.19b 4.59 ±3.98b 7.16 ±3.18
pvalues 0.0366 0.0011 0.0003 0.0011 0.0169 0.0797
n 25 400 100 100 100 100
d
13
C Douglas fir -31.25 ±0.11c -26.43 ±0.57c -26.14 ±0.43c -26.57 ±0.55c -26.50 ±0.69c -26.52 ±0.65c
[%] Pine -32.19 ±0.34b -27.49 ±0.52b -27.06 ±0.21b -27.47 ±0.57b -27.61 ±0.43b -27.83 ±0.56ab
Beech -33.17 ±0.25a -27.63 ±0.41b -27.64 ±0.43b -27.89 ±0.29b -27.67 ±0.49b -27.32 ±0.28bc
Oak -32.06 ±0.46b -28.89 ±0.56a -28.88 ±0.58a -29.25 ±0.41a -28.90 ±0.63a -28.51 ±0.50a
pvalues 0.0010 < 0.0001 < 0.0001 < 0.0001 0.0001 0.0001
n 25 100 25 25 25 25
d
15
N Douglas fir -5.22 ±0.65c -2.54 ±1.39c -2.19 ±1.76c -2.13 ±1.62c -2.69 ±1.19b -3.18 ±1.02b
[%] Pine -8.25 ±0.58ab -5.91 ±0.82b -6.05 ±0.66ab -5.45 ±0.79a -6.08 ±1.01a -6.07 ±0.89a
Beech -7.79 ±0.22b -4.10 ±1.57b -4.89 ±0.40bc -3.65 ±2.33bc -3.19 ±1.88ab -4.66 ±0.45ab
Oak -8.74 ±0.37a -5.42 ±0.98a -6.31 ±0.58a -4.71 ±0.73ab -5.63 ±1.06a -5.02 ±0.87a
pvalues < 0.0001 < 0.0001 0.0014 0.0126 0.0052 0.0005
n 25 100 25 25 25 25
Root properties are given on horizon level as well as sum (biomass) and on average (d
13
C, d
15
N) of roots in the forest floor and mineral soil, irrespective of soil horizons (‘‘Roots
general’’). Values are mean ±SD. Differences between tree species are marked by different letters. Significant pvalues (\0.05) are highlighted in bold font style. The total
number of samples (n) per parameter and substrate comprises all four tree species
123
Biogeochemistry (2020) 151:203–220 209
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

b
d13C
values and e
soil
15
N and the contribution
of litterfall and root inputs
The b
d13C
values for beech (-1.14) were significantly
more negative compared to pine (-0.64) and Douglas
fir (-0.81) by a factor of 1.8, 1.4 respectively
(Fig. 4a). For the oak stands intermediate b
d13C
values
(-0.87) were determined. Values for e
soil
15
N differed
up to a factor of 1.7 with significantly higher values
under beech (7.02%) and pine (6.52%) compared to
oak (4.76%) and Douglas fir (4.05%) (Fig. 4b).
Apparently, b
d13C
values differed systematically
between coniferous and deciduous species, while
e
soil
15
N depended more on individual tree species.
Using multiple linear regression analyses, the
impact of litterfall and root properties on both indices,
b
d13C
and e
soil
15
N, was assessed. In total, 49% and
74% of the variability in b
d13C
and e
soil
15
N was
represented by the explaining variables (Table 3).
Higher b
d13C
values were associated with litterfall that
was characterized by higher d
13
C values and lower
d
15
N values. Furthermore, root C:N played a signif-
icant role for b
d13C
. Litterfall with lower C:N ratios
and more negative d
15
N values were related to higher
e
soil
15
N. Additionally, higher root d
13
C values and
lower O:C ratios of roots were associated with higher
e
soil
15
N.
Fig. 2 Depth gradients of C (a), d
13
C(b), N (c) and d
15
N
(d) from the OL horizon to 10–30 cm. Coniferous species
Douglas fir (‘‘D’’) and pine (‘‘P’’) are presented by black
symbols and the deciduous species beech (‘‘B’’) and oak (‘‘O’’ )
by grey symbols. Significant differences between tree species
are marked with ‘‘*’’ (p\0.05), ‘‘**’’ (p\0.01), ‘‘***’’
(p\0.001). Detailed information about statistical differences
can be found in Table S4
Table 2 Results of the regression analyses between isotopic
and stoichiometric ratios
Function R
2
p values
d
13
C vs. C/N
Douglas fir y = -0.181 ln(x) -1.763 0.38 0.0003
Pine y = -0.252 ln(x) -3.745 0.32 0.0011
Beech y = -0.191 ln(x) -2.295 0.56 < 0.0001
Oak y = -0.248 ln(x) -3.740 0.30 0.0018
d
13
C vs. O/C
Douglas fir y = 0.538 ln(x) ?14.472 0.55 < 0.0001
Pine y = 0.595 ln(x) ?16.336 0.51 < 0.0001
Beech y = 0.550 ln(x) ?15.463 0.76 < 0.0001
Oak y = 0.402 ln(x) ?11.131 0.45 < 0.0001
d
15
N vs. C/N
Douglas fir y = -0.141 ln(x) ?2.889 0.79 < 0.0001
Pine y = -0.124 ln(x) ?2.796 0.85 < 0.0001
Beech y = -0.105 ln(x) ?2.772 0.88 < 0.0001
Oak y = -0.180 ln(x) ?2..662 0.75 < 0.0001
d
15
N vs. O/C
Douglas fir y = 0.363 ln(x) ?0.521 0.85 < 0.0001
Pine y = 0.234 ln(x) ?0.711 0.86 < 0.0001
Beech y = 0.263 ln(x) ?0.763 0.90 < 0.0001
Oak y = 0.244 ln(x) ?0.602 0.80 < 0.0001
Significant pvalues (\0.05) are highlighted in bold font style
123
210 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Fig. 3 Relationship between d
15
N and the molar ratios of C:N
and O:C. Douglas fir (‘‘D’’) and pine (‘‘P’’) are presented by blue
and green symbols, beech (‘‘B’’) and oak (‘‘O’’) are represented
by yellow and red symbols. Detailed information about statistics
of the logarithmic and linear relationships can be found in
Table 2and Table S5
Fig. 4 Boxplots of b
d13C
(slopes of the linear regression between d
13
C and log C) (a) and e
soil
15
N (SOM
15
N enrichment from OL to
10–30 cm) (b) of investigated forest stands. Black rhombuses represent mean values
Table 3 Final models with
explaining variables of the
multiple linear regression
analysis for the
determination of factors
influencing b
d13C
and
e
soil
15
N
Significant pvalues (\
0.05) are highlighted in
bold font style
Model parameter R
2
pvalues
b
d13C
Litterfall d
13
C?litterfall d
15
N?root C:N 0.46 0.0046
e
soil
15
N Litterfall C:N ?litterfall d
15
N?root O:C ?root d
13
C 0.74 < 0.0001
Proxy Explaining variables Coefficients pvalues
b
d13C
Intercept 5.63 0.0245
Litterfall d
13
C 0.21 0.0131
Litterfall d
15
N-0.09 0.0337
Root C:N -0.01 0.0114
e
soil
15
N Intercept 42.74 0.0002
Litterfall C:N -0.04 0.0361
Litterfall d
15
N-1.14 < 0.0001
Root O:C -18.61 0.0086
Root d
13
C 1.19 0.0089
123
Biogeochemistry (2020) 151:203–220 211
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Discussion
Isotopic signatures of litter inputs
In all investigated forest stands roots were consistently
enriched in
13
C and
15
N compared to the litterfall
(Table 1). Several post-photosynthetic allocation
mechanisms can lead to an enrichment of
13
Cin
heterotrophic plant organs compared to leaves (Cer-
nusak et al. 2009). For example, a greater allocation of
depleted C to lignin and lipid pools and an export of
less depleted carbohydrates to roots result in an
enrichment of
13
C in belowground organs (Hobbie
and Werner 2004; Badeck et al. 2005). The observed
significant impact of tree species on root and litterfall
d
13
C is caused by a complex interplay of physiological
differences between the tree species and their response
to environmental conditions, which has been thor-
oughly reviewed by Dawson et al. (2002). The higher
d
13
C values of the here investigated coniferous species
in comparison to deciduous trees (Table 1) are mainly
caused by a higher intrinsic water-use-efficiency,
lower stomatal conductance and lower photosynthetic
rates (Brooks et al. 1997). It must be noted that the
d
13
C of different plant parts varies on diurnal, seasonal
and annual to interannual time scales (Bru
¨ggemann
et al. 2011). Here we use the d
13
C of the annual litter
inputs as reference points to evaluate the decomposi-
tion of OM along soil profiles (Bowling et al. 2008).
The generally lower d
15
N values of litterfall
compared to roots are in line with findings of other
studies (Ho
¨gberg et al. 1996; Templer et al. 2007).
This pattern can be assigned to fractionation during N
transformation and transport within the plant that leads
to an assimilation of
15
N-depleted N in leaves and
15
N-
enriched N in roots (Pardo et al. 2013). Moreover, the
formation of mycorrhizal symbioses is one of the most
important factors influencing the d
15
N signature of
leaves. The here investigated tree species Douglas fir,
pine, beech and oak are well known to form symbioses
with ectomycorrhizal (EM) fungi (Wang and Qiu
2006).
15
N-enriched N compounds are preferentially
retained by the fungal biomass, while
15
N-depleted N
compounds are transported to their host plant (Craine
et al. 2009). The biggest difference between root and
litterfall d
15
N was determined for beech
(3.69 ±0.96%) followed by oak (3.33 ±0.52%).
This range is in agreement with differences of *4%
observed by Hobbie and Colpaert (2003). According
to them, the amount of ectomycorrhizal mass included
with the roots also determines the enrichment of roots
in
15
N compared to foliar tissues. The threefold higher
root biomass of beech and oak compared to Douglas fir
and pine (Table 1) can therefore be responsible for the
highest differences between the plant organs at these
stands. Analyzing the abundance of mycorrhizal fungi
in symbioses with the investigated tree stands was
beyond the scope of the study. However, it is reported
that the EM fungal biomass does not vary significantly
between beech and conifers in temperate forests but
the mechanisms behind the regulation of EM fungal
biomass are highly complex (Awad et al. 2019).
Additional to mycorrhizal associations, the variability
in plant d
15
N depends on the form of soil N that plants
predominantly acquire (Vallano and Sparks 2013).
Denitrification and nitrification both discriminate
against
15
N because
15
N-depleted nitrate can be
leached from the soil, resulting in
15
N-enrichment of
the remaining N that can be taken up by plants (Hobbie
and Ho
¨gberg 2012). High nitrate concentrations of
soils under Douglas fir (Zeller et al. 2019) can account
for the significantly highest litterfall d
15
N values at the
study site ‘Sophienho
¨he’. Beech, with the second
highest d
15
N values for litter inputs, is recognized as a
tree species that promotes nitrification in soils (An-
drianarisoa et al. 2010). However, a more profound
investigation of specific N cycling processes in the
plant-soil system that potentially influence the natural
abundance of d
15
N is beyond the scope of this study.
Depth profiles of d
13
C and d
15
N and their
relationship to stoichiometry patterns
Within 35 years after afforestation distinct depth
profiles of d
13
C developed in all investigated forest
stands confirming findings of Brunn et al. (2017), who
demonstrated that three decades after afforestation are
sufficient to yield such profiles. The gradients from OL
to OH in our investigated forest stands were charac-
terized by a decrease or at least no alteration of d
13
C
(Fig. 2b). Within the early stages of OM decomposi-
tion water-soluble substances and non-lignified car-
bohydrates are degraded, while the proportion of
lignin residually increases (Berg 2008; Osono et al.
2008). Lignin is characterized by lower d
13
C values
compared to bulk foliar d
13
C, while cellulose and
sugars are characterized by higher values (Bowling
et al. 2008). Therefore, a selective preservation of
123
212 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

lignin, and lignin building blocks, respectively
(Sua
´rez-Abelenda et al. 2015), which cannot be
attacked by the vast majority of decomposers, can
lead to the depletion of
13
C downwards through the
organic horizons under Douglas fir, pine and oak. No
significant depletion of
13
C was found under beech. It
appears that beech maybe belongs to the group of tree
species, where the carbohydrate-dominated early
stage of litter decomposition is so marginal that it
has no measurable impact on d
13
C values (Berg and
McClaugherty 2014). The fact that the sampling
campaign was in April and the litterfall predominately
occurred in October and November corroborates the
assumption. The time difference between litterfall and
organic-layer sampling can also be responsible for the
high extent of
13
C enrichment from litterfall to OL
material. In the mineral soil horizons, however, d
13
C
of bulk SOM increased with increasing depth. A
relevant contribution of atmospheric
13
C-depleted
CO
2
(Francey et al. 1999), to OM at the soil surface
can be excluded because the 35 years old afforested
sites are rather young. Instead,
13
C-depleted litter from
aboveground plant materials accumulates at the soil
surface, while the contribution of OM that derives
from
13
C-enriched roots to SOM formation increases
with soil depth (Bird et al. 2003). Correspondingly, in
our study roots were on average higher with
13
Cby
4.56 ±0.99%compared to the litterfall. Furthermore,
the kinetic fractionation of C isotopes during the
maturation of SOM leads to an enrichment of
13
C with
increasing depth (Wynn et al. 2006). Within the
microbial metabolism of C sources preferentially
13
C-
depleted CO
2
is respired by microorganisms, while the
remaining SOM including the soil microbial biomass
becomes enriched in
13
C (Werth and Kuzyakov 2010).
Thus, microorganisms fractionate during the C assim-
ilation and/or preferentially use
13
C-enriched sub-
strates (Schwartz et al. 2007). Especially in mineral
soils of forests
13
C-enriched microbial-derived OM
has a larger share of bulk SOM d
13
C values than lignin
or aliphatic biopolymers (Du
¨mig et al. 2013).
Throughout the soil profile, d
13
C of SOM was affected
by tree identity with consistently highest values in
Douglas fir stands and lowest values in oak stands. In
contrast, Marty et al. (2015) found a negative impact
of the percentage of conifers in Canadian forests on
d
13
C values in mineral horizons. They assume that this
was caused by lower microbial activity and/or lower
SOM degradation at sites dominated by conifers. Yet,
in our study the differences in the SOM d
13
C values
among tree species reflect the isotopic signatures of
the OL horizon that in turn strongly correlated with
litterfall d
13
C. This explains why OM under Douglas
fir with highest d
13
C values in litterfall and roots
exhibited the highest d
13
C values throughout the soil
profile, while they were lowest under oak.
In coincidence with the
13
C enrichment with
increasing soil depth, gradients of d
15
N from the OL
down to the 10–30 cm layer of mineral soil had
developed in all forest stands during 35 years of
afforestation (Fig. 2d). The depth distribution of SOM
d
15
N mainly results from an interplay of input
signatures and losses that occur during decomposition
processes (Craine et al. 2015). The accumulation of
15
N-depleted plant litter on the soil surface determines
the gradient from the significantly lower d
15
N values
of forest floor horizons to the
15
N-enriched mineral
soil. Thus, the highest d
15
N values in the forest floor
horizons under Douglas fir reflect the highest d
15
N
values of the litterfall and roots of all tree species that
in turn were determined by the nitrate concentrations
of Douglas fir soils (Zeller et al. 2019). The lowest
forest floor d
15
N values that were observed in the pine
stand are in accordance with other studies revealing
that conifer-dominated sites were
15
N-depleted com-
pared to deciduous species (Pardo et al. 2007). In
contrast to the d
13
C depth gradients, d
15
N increased
consistently throughout the soil profile following a
curve that is typical for N-limited forest ecosystems
dominated by EM fungi (Hobbie and Ouimette 2009).
The clearly higher d
15
N values under beech and
Douglas fir compared to oak and pine are in accor-
dance with observations made in a common garden
experiment in Poland (Angst et al. 2019). With
increasing depth and ongoing decomposition, SOM
becomes preferentially
15
N-enriched due to microbial
activity coupled with an increasing proportion of
15
N-
enriched microbial derived compounds (Lerch et al.
2011). The individual SOM d
15
N depth gradients of
tree species converged in the upper two mineral soil
horizons (0–5, 5–10 cm) and diverged again with
increasing depth implying that SOM turnover differed
under the influence of tree species. Additionally, tree
species and their mycorrhizal symbionts, respectively,
also contributes to the d
15
N depth profiles by their N
uptake from soil (Handley and Raven 1992; Callesen
et al. 2013). The type of mycorrhizal association
mainly drives the form of N acquisition of temperate
123
Biogeochemistry (2020) 151:203–220 213
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

tree species (Liese et al. 2017) and leads to differences
in
15
N enrichments in soil profiles between EM and
arbuscular mycorrhizal fungi dominated systems
(Hobbie and Ho
¨gberg 2012). The here investigated
tree species are all dominated by EM fungi with
similar fungal biomasses (Wang and Qiu 2006; Awad
et al. 2019) and thus, the N transfer from the soil to the
host plant is presumably not significantly different
between tree species. Anyhow, the turnover of SOM
and the N uptake by plants are highly interrelated and
therefore both mechanisms will have contributed to
the d
15
N depth profiles.
The negative relationship between d
15
N and soil
C:N ratio may result from increasing loss of
15
N-
depleted N in the form of nitrate leaching or denitri-
fication as consequence of decreasing N retention
(Marty et al. 2019). However, this option is likely
subordinate because N-limited temperate forests are
characterized by a largely closed internal N cycle,
where N-losses are generally low due to a high
competition for this growth-limiting resource (Ren-
nenberg et al. 2009). Rather, the relationship of d
15
N
to the soil C:N is best explained by the increase in OM
decomposition with increasing soil depth. The rela-
tionship between d
13
C and soil C:N ratio, that is also
negative, supports this assumption (Baisden et al.
2002). The well-known decline of the C:N ratio with
increasing depth (Marı
´n-Spiotta et al. 2014) is mostly
attributed to OM decay because substrate that accu-
mulates at the soil surface has significantly higher C:N
ratios compared to decomposers and their products
(Manzoni et al. 2010; Paul 2016). The slopes of the
specific regression lines (Fig. 3a) were more driven by
the enrichment of
15
N with increasing depth than by
the C:N ratio. Steeper slopes in beech and pine stands
were associated with the significantly higher e
soil
15
N
values of beech and pine compared to oak and Douglas
fir. Nonetheless, the significant relationship between
d
15
N and the C:N ratio emphasizes the potential of
15
N
depth gradients as proxy for OM decay. Kramer et al.
(2017) found that changes in organo-mineral associ-
ations can drive depth trends of C:N and d
15
N more
than the microbial decay. However, this effect was
largely reduced, since forest stands with uniform
mineral phase were investigated in this study. Fur-
thermore, the O:C ratio that represents the state of
chemical oxidation (Fan et al. 2018) of SOM was also
significantly related to d
15
N and d
13
C throughout the
soil profile. Oxidation is accompanied with microbial
breakdown and depolymerization of plant residues
followed by assimilation of C in microbial biomass as
well as mineralization at the same time (Lehmann and
Kleber 2015). Thus, with increasing depth the rise of
the O:C ratio indicated the progressive oxidative
degradation of OM and correlated with the enrichment
of
15
N and
13
C in SOM. All this led to the assumption
that the depth trends of SOM d
15
N and d
13
C resulted
mainly from the decomposition of OM.
b
d13C
values and e
soil
15
N and the contribution
of litterfall and root inputs
In well-drained forest soils, like our study site
‘Sophienho
¨he’, the linear regression function of d
13
C
and the logarithm of SOC with soil depth, termed as
b
d13C
value, is a suitable indicator of isotopic
fractionation during decomposition (Brunn et al.
2014). Physical soil mixing processes that could also
have a contribution to the isotopic fractionation with
soil depth (Acton et al. 2013) can be excluded because
during field surveys no earthworms or signs of
significant bioturbation processes were found (Lorenz
and Thiele-Bruhn 2019). However, steeper regression
slopes, and more negative b
d13C
values respectively,
indicate higher rates of
13
C enrichment through the
soil depth profile and enhanced organic matter turn-
over (Garten 2006; Wang et al. 2018). Tree species
had a significant effect on b
d13C
values. The most
negative b
d13C
values and therefore the highest rates of
SOM turnover were determined in beech forest stands,
while reduced SOM turnover at coniferous sites was
indicated by less negative b
d13C
values. This is in
accordance with the view that turnover rates, espe-
cially in the early-stage of decomposition, of decid-
uous species litter are generally higher compared to
conifers (Augusto et al. 2015 and references in there).
Long-term studies ([10 years) suggest that there are
also significant differences in the remaining masses
after decomposition between tree species (Harmon
et al. 2009; Prescott 2010). This can be addressed to
significantly higher N contents and lower C:N ratios in
the litterfall of beech and oak (Table S3), because it is
well documented that these parameters correlate well
with decomposition (Fernandez et al. 2003; Laganie
`re
et al. 2010; Vesterdal et al. 2012). Our findings are
confirmed by other studies revealing that more neg-
ative b
d13C
values were related to higher N contents
and lower C:N ratios of litterfall (Garten et al. 2000;
123
214 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Garten 2006; Wang et al. 2015). The multiple linear
regression analysis, which included different quantity
and quality properties of the litter inputs, pointed out
that the isotopic signatures of litterfall and root C:N
account for nearly half of the variation (46%) in b
d13C
values. This implies on the one hand that the initial
isotopic composition of the aboveground litter plays a
crucial role, for evaluating the
13
C enrichment in soil
depth profiles in the context of SOM turnover.
Camino-Serrano et al. (2019) figured out that litter
d
13
C is the key to predict and model d
13
C depth
profiles. On the other hand, the C and N stoichiometry
of root biomass seems to be of high importance for
b
d13C
values in forest soils. Belowground inputs are
still less researched but knowledge is growing that
these inputs have a significantly contribution to OM
formation (Angst et al. 2018; Poirier et al. 2018). For
example, Kramer et al. (2010) demonstrated that root-
derived C is the major ([60%) source of C for
microbes in temperate deciduous forest soils, while
also in boreal forest soils 50 to 70% of the stored C
derives from roots and root-associated microorgan-
isms (Clemmensen et al. 2013). However, 54% of the
variation in b
d13C
values cannot be explained with the
here investigated properties of litterfall inputs and root
material suggesting that SOM decomposition depends
additionally on other factors.
The use of bulk SOM d
15
N as proxy for turnover
and stabilization of SOM bears an uncertainty due to
factors like the variability of the initial abundance of
15
N in litter inputs (Ho
¨gberg 1997). Thus, we evalu-
ated soil enrichment factors (e
soil
15
N) to level out the
different isotopic signatures of litter inputs. Like the
b
d13C
values, e
soil
15
N values were significantly
affected by tree species but the differences between
tree species are not consistent for e
soil
15
N and b
d13C
values. If mainly microbial turnover of SOM would
drive both parameters in similar trajectories, this
would result in a negative relationship between both
indices. This applies for Douglas fir, oak and in
particular for beech with oppositely high or low b
d13C
and e
soil
15
N values, respectively (Fig. 4). However,
soils under pine were characterized by highest b
d13C
as
well as high e
soil
15
N values. This indicates that in pine
stands
13
C and
15
N enrichments with soil depth are
decoupled from each other and not systematically
interlinked. Nel et al. (2018) reported on a general link
between d
13
C and d
15
N on global scale and with soil
depth but they also mentioned that this link varied with
local influences from biota, disturbance and environ-
mental conditions. Multiple linear regression analysis
revealed that litterfall C:N and d
15
N as well as root
O:C and d
13
C account for 74% of the variability in
e
soil
15
N. This highlights the importance of litterfall and
root inputs for the
15
N enrichment with soil depth. The
general view that the higher decomposition rates of
litter with high N content and low C:N ratio (Garten
et al. 2000) are associated with both, high
15
N and
13
C
enrichment does not match with the finding of high
15
N enrichment in soils under pine. This must be
caused by other processes. The high C:N ratio of the
N-poor litterfall (Table S3) suggests that the N supply
in the upper 30 cm of soil in the pine stands is limited.
The significantly lowest root biomasses of the typical
deep rooting tap root system (Burylo et al. 2011)
combined with its low turnover rates (Yuan and Chen
2010) will also provide not much N. Under such
N-limited conditions EM fungi are able to oxidize OM
primarily as N source rather than as a source of
metabolic C (Lindahl and Tunlid 2015). Thus, EM
fungi in the pine stands can compete directly with
decomposers for soil N resources and exacerbate the
N-limitation of free-living decomposers (Averill
2016). Therefore, the soil microbial community could
adapt their N-utilization strategy to an efficient re-use
of organic N derived from their own bio- and
necromass. The multiple recycling of these N sources
will lead to an ongoing enrichment of
15
N without
higher rates of decomposition and could end up in the
discrepancy between b
d13C
and e
soil
15
N values. Fur-
thermore, it has been reported that under conditions
with low C availability, N-containing organic com-
pounds can be primarily used as source of C and
energy. Consequently, dissimilated
15
N-depleted N
will be exported and the microbial cell gets enriched
relative to its source (Dijkstra et al. 2008). Conse-
quently, both mechanisms, the effective recycling of
microbial derived N under N-limited conditions as
well as the dissimilation of
15
N-enriched N, when the
relative availability of C is low, could lead to high
e
soil
15
N and low b
d13C
values in pine stands.
Conclusion
The post-mining site ‘‘Sophienho
¨he’’ represented a
suitable site to characterize the influence of tree
species on the natural abundance of
13
C and
15
N in soil
123
Biogeochemistry (2020) 151:203–220 215
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

depth gradients. As an advantage to many common
garden experiments, an interference from old C
sources originating from former land use was negli-
gible. Additionally, 35 years after the afforestation
were sufficient to generate tree species-specific depth
gradients. Hence, evidence was provided that differ-
ences in isotopic signatures of SOM originated from
the input of plant litter and its decomposition products.
The significantly different d
13
C and d
15
N values in
the OM of forest floor and mineral soil reflected the
signatures of the litter inputs (litterfall and roots) that
were tree species specific. Along the soil profile, both
isotopes were significantly related to the C:N and O:C
ratio indicating that the enrichment of
13
C and
15
N
with increasing soil depth is driven by processes that
presumably can be assigned to microbial decomposi-
tion of OM. Consequently, when
13
C and
15
N of bulk
SOM are used to evaluate decomposition and stabi-
lization of OM, the isotopic signatures of litter inputs
should be considered as well. Differences in b
d13C
values indicated different turnover of SOM between
tree species with higher decomposition rates in
deciduous forest stands compared to conifers. The
quality of litterfall and root inputs (N content, C:N,
O:C ratio) as well as the initial isotopic signatures of
litterfall contributed to the regulation of OM decom-
position. Yet, 54% of the variance in b
d13C
, and 26% in
e
soil
15
N respectively, cannot be explained with the here
investigated litterfall and root properties showing that
SOM decomposition depends additionally on other—
presumably microbial driven—factors. The corre-
spondence of e
soil
15
N values with b
d13C
values in
three of the four investigated forest stands (Douglas
fir, beech, oak) suggests that the
13
C and
15
N
enrichment with increasing depth followed similar
principles. However, the conditions under pine did not
follow the systematic link between
13
C and
15
N
enrichment. This is presumably due to specific N
cycling mechanisms mediated by microorganisms that
were adapted to conditions of limited N availability
and the relatively low availability of C.
It is concluded that typical pattern of
13
C and
15
N
enrichment with increasing soil depth are due to
maturation and ongoing turnover of SOM. However,
under the influence of tree species the enrichment of
both isotopes did not follow similar trajectories in
general because of microorganisms that can create
specific utilization strategies depending on the litter
quality. It was possible to obtain this finding by
combining stable isotope analysis with the classical
determination of stoichiometry ratios (C:N, O:C).
Acknowledgements The authors thank the colleagues of the
Soil Science Department of Trier University, P. Ziegler and M.
Ortner, and the students, K. Becker, L. von Drathen, S. Stein and
L. Schneider, for assistance during laboratory and field work.
We also thank the certified facility in Functional Ecology (PTEF
OC 081) from UMR 1137 EEF and UR 1138 BEF in the research
centre INRAE Grand-Est–Nancy, and in particular S. Moutama,
J. Ph. Gallais for sample preparation and weighting and C.
Hossann, for performing/supervising isotopic analyses. Many
thanks to Oliver Brendel (INRA, Champenoux, France) for
suggestions in the data interpretation. This research did not
receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors. The UR BEF is supported
by the French National Research Agency through the Cluster of
Excellence ARBRE (ANR-11-LABX-0002-01). Additional
support was provided by the mobile lab (M-POETE) of
ANAEE-France. ANAEE-France is an infrastructure from the
French Investment for the Future (Investissements d’Avenir)
program, overseen by the French National Research Agency
(ANR-11-INBS-0001).
Funding Open Access funding enabled and organized by
Projekt DEAL..
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any med-
ium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative
Commons licence, and indicate if changes were made. The
images or other third party material in this article are included in
the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
References
Acton P, Fox J, Campbell E, Rowe H, Wilkinson M (2013)
Carbon isotopes for estimating soil decomposition and
physical mixing in well-drained forest soils. J Geophys Res
Biogeosci 118(4):1532–1545. https://doi.org/10.1002/
2013JG002400
Andrianarisoa KS, Zeller B, Poly F, Siegenfuhr H, Bienaime
´S,
Ranger J, Dambrine E (2010) Control of nitrification by
tree species in a common-garden experiment. Ecosystems
123
216 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

13:1171–1187. https://doi.org/10.1007/s10021-010-9390-
x
Angst G, Messinger J, Greiner M, Ha
¨usler W, Hertel D, Kirfel
K, Ko
¨gel-Knabner I, Leuschner C, Rethemeyer J, Mueller
CW (2018) Soil organic carbon stocks in topsoil and sub-
soil controlled by parent material, carbon input in the rhi-
zosphere, and microbial-derived compounds. Soil Biol
Biochem 122:19–30. https://doi.org/10.1016/j.soilbio.
2018.03.026
Angst G, Mueller KE, Eissenstat DM, Trumbore S, Freeman
KH, Hobbie SE, Chorover J, Olekysn J, Reich PB, Mueller
CW (2019) Soil organic carbon stability in forests: distinct
effects of tree species identity and traits. Glob Change Biol
25:1529–1546. https://doi.org/10.1111/gcb.14548
Augusto L, De Schrijver A, Vesterdal L, Smolander A, Prescott
C, Ranger J (2015) Influences of evergreen gymnosperm
and deciduous angiosperm tree species on the functioning
of temperate and boreal forests. Biol Rev 90:444–466.
https://doi.org/10.1111/brv.12119
Averill C (2016) Slowed decomposition in ectomycorrhizal
ecosystems is independent of plant chemistry. Soil Biol
Biochem 102:52–54. https://doi.org/10.1016/j.soilbio.
2016.08.003
Awad A, Majcherczyk A, Schall P, Schro
¨ter K, Scho
¨ning I,
Schrumpf M, Ehbrecht M, Boch S, Kahl T, Bauhus J,
Seidel D, Ammer C, Fischer M, Ku
¨es U, Pena R (2019)
Ectomycorrhizal and saprotrophic soil fungal biomass are
driven by different factors and vary among broadleaf and
coniferous temperate forests. Soil Biol Biochem 131:9–18.
https://doi.org/10.1016/j.soilbio.2018.12.014
Badeck FW, Tcherkez G, Nogue
´s S, Piel C, Gashghaie J (2005)
Post-photosynthetic fractionation of stable carbon isotopes
between plant organs—a widespread phenomenon. Rapid
Commun Mass Spectrom 19:1381–1391. https://doi.org/
10.1002/rcm.1912
Baisden WT, Amundson R, Cook AC, Brenner DL (2002)
Turnover and storage of C and N in five density fractions
from California annual grassland surface soils. Glob Bio-
geochem Cycles 16:64-1–64-16. https://doi.org/10.1029/
2001GB001822
Balesdent J, Mariotti A, Guillet B (1987) Natural
13
C abundance
as a tracer for studies of soil organic matter dynamics. Soil
Biol Biochem 19:25–30. https://doi.org/10.1016/0038-
0717(87)90120-9
Balesdent J, Basile-Doelsch I, Chadoeuf J, Cornu S, Derrien D,
Fekiacova Z, Hatte
´C (2018) Atmosphere–soil carbon
transfer as a function of soil depth. Nature 559:599–602.
https://doi.org/10.1038/s41586-018-0328-3
Berg B (2008) Litter decomposition and organic matter turnover
in northern forest soils. For Ecol Manag 133:13–22. https://
doi.org/10.1016/S0378-1127(99)00294-7
Berg B, McGlaugherty C (2014) Plant litter. Decomposition,
humus formation, carbon sequestration, 3rd edn. Springer,
Berlin
Beyer L, Deslis K, Vogt B (1998) Estimation of soil organic
matter composition according to a simple thermoanalytical
approach. Commun Soil Sci Plant Anal 29:1277–1297.
https://doi.org/10.1080/00103629809370026
Billings SA, Richter D (2006) Changes in stable isotopic sig-
natures of soil nitrogen and carbon during 40 years of
forest development. Oecologia 148:325–333. https://doi.
org/10.1007/s00442-006-0366-7
Bird MI, Kracht O, Derrien D, Zhou Y (2003) The effect of soil
texture and roots on the stable isotope composition of soil
organic matter. Aust J Soil Res 41:77–94. https://doi.org/
10.1071/SR02044
Bostro
¨m B, Comstedt D, Ekblad A (2007) Isotope fractionation
and
13
C enrichment in soil profiles during the decomposi-
tion of soil organic matter. Oecologia 153:89–98. https://
doi.org/10.1007/s00442-007-0700-8
Bowling DR, Pataki DE, Randerson JT (2008) Carbon isotopes
in terrestrial ecosystem pools and CO
2
fluxes. New Phytol
178:24–40. https://doi.org/10.1111/j.1469-8137.2007.
02342.x
Brooks JR, Flanagan LB, Buchmann N, Ehleringer JR (1997)
Carbon isotope composition of boreal plants: functional
grouping of life forms. Oecologia 110:301–311. https://
doi.org/10.1007/s004420050163
Bru
¨ggemann N, Gessler A, Kayler Z, Keel Z, Badeck F, Barthel
M, Boeckx P, Buchmann N, Brugnoli E, Esperschu
¨tz J,
Gavrichkova O, Ghashghaie J, Gomez-Casanovas N,
Keitel C, Knowhl A, Kuptz D, Palacio S, Salmon Y,
Uchida Y, Bahn M (2011) Carbon allocation and carbon
isotope fluxes in the plant-soil-atmosphere continuum: a
review. Biogeosciences 8:3457–3489. https://doi.org/10.
5194/bg-8-3457-2011
Brunn M, Spielvogel S, Sauer T, Oelmann Y (2014) Tempera-
ture and precipitation effects on d
13
C depth profiles in
SOM under temperate beech forests. Geoderma
235–236:146–153. https://doi.org/10.1016/j.geoderma.
2014.07.007
Brunn M, Brodbeck S, Oelmann Y (2017) Three decades fol-
lowing afforestation are sufficient to yield d
13
C depth
profiles. J Plant Nutr Soil Sc 180:643–647. https://doi.org/
10.1002/jpln.201700015
Burylo M, Hudek C, Rey F (2011) Soil reinforcement by the
roots of six dominant species on eroded mountainous marly
slopes (Southern Alps, France). CATENA 84:70–78.
https://doi.org/10.1016/j.catena.2010.09.007
Callesen I, Nilsson LO, Schmidt IK, Vesterdal L, Ambus P,
Christiansen JR, Ho
¨gberg P, Gundersen P (2013) The
natural abundance of
15
N in litter and soil profiles under six
temperate tree species: N cycling depends on tree species
traits and site fertility. Plant Soil 368:375–392. https://doi.
org/10.1007/s11104-012-1515-x
Camino-Serrano M, Tifafi M, Balesdent J, Hatte
´C, Pen
˜uelas J,
Cornu S, Guenet B (2019) Including stable carbon isotopes
to evaluate the dynamics of soil carbon in the land-surface
model ORCHIDEE. J Adv Model Earth Syst
11:3650–3669. https://doi.org/10.1029/2018MS001392
Cernusak LA, Tcherkez G, Keitel C, Cornwell WK, Santiago
LS, Knohl A, Barbour MM, Williams DG, Reich PB,
Ellsworth DS, Dawson TE, Griffiths HG, Farquhar GD,
Wright IJ (2009) Why are non-photosynthetic tissues
generally
13
C enriched compared with leaves in C3 plants?
Review and synthesis of current hypotheses. Funct Plant
Biol 36:199–213. https://doi.org/10.1071/FP08216
Clemmensen KE, Bahr A, Ovaskainen O, Dahlberg A, Ekblad
A, Wallander H, Stenlid J, Finlay RD, Wardle DA, Lindahl
BD (2013) Roots and associated fungi drive long-term
123
Biogeochemistry (2020) 151:203–220 217
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

carbon sequestration in boreal forest. Science
339:1615–1618. https://doi.org/10.1126/science.1231923
Cools N, Vesterdal L, De Vos B, Vanguelova E, Hansen K
(2014) Tree species is the major factor explaining C:N
ratios in European forest soils. For Ecol Manag 311:3–16.
https://doi.org/10.1016/j.foreco.2013.06.047
Craine JM, Elmore AJ, Aidar MPM, Bustamante M, Dawson
TE, Hobbie EA, Kahmen A, Mack MC, McLauchlan KK,
Michelsen A, Nardoto GB, Pardo LH, Pen
˜uela J, Reich PB,
Schuur G, Stock WD, Templer PH, Virginia RA, Welker
JM, Wright IJ (2009) Global patterns of foliar nitrogen
isotopes and their relationships with climate, mycorrhizal
fungi, foliar nutrient concentrations, and nitrogen avail-
ability. New Phytol 183:980–992. https://doi.org/10.1111/
j.1469-8137.2009.02917.x
Craine JM, Brookshire ENJ, Cramer MD, Hasselquist NJ, Koba
K, Marin-Spiotta E, Wang L (2015) Ecological interpre-
tations of nitrogen isotope ratios of terrestrial plants and
soils. Plant Soil 396:1–26. https://doi.org/10.1007/s11104-
015-2542-1
Dawson TE, Mambelli S, Plamboeck AH, Templer PH, Tu KP
(2002) Stable isotopes in plant ecology. Annu Rev Ecol
Syst 33:507–559. https://doi.org/10.1146/annurev.ecolsys.
33.020602.095451
Dijkstra P, Ishizu A, Doucett R, Hart SC, Schwartz E, Menyailo
OV, Hungate BA (2006)
13
C and
15
N natural abundance of
the soil microbial biomass. Soil Biol Biochem
38:3257–3266. https://doi.org/10.1016/j.soilbio.2006.04.
005
Dijkstra P, LaViolette CM, Coyle JS, Doucett RR, Schwartz E,
Hart SC, Hungate BA (2008)
15
N enrichment as an inte-
grator of the effects of C and N on microbial metabolism
and ecosystem function. Ecol Lett 11:389–397. https://doi.
org/10.1111/j.1461-0248.2008.01154.x
Du
¨mig A, Rumpel C, Dignac MF, Ko
¨gel-Knabner I (2013) The
role of lignin for the d
13
C signature in C4 grassland and C3
forest soils. Soil Biol Biochem 57:1–13. https://doi.org/10.
1016/j.soilbio.2012.06.018
Ehleringer JR, Buchmann N, Flanagan LB (2000) Carbon iso-
tope ratios in belowground carbon cycle processes. Ecol
Appl 10(2):412–422
Emmet BA, Kjønaas OJ, Gundersen P, Koopmans C, Tietema
A, Sleep D (1998) Natural abundance of
15
N in forests
across a nitrogen deposition gradient. For Ecol Manag
101:9–18. https://doi.org/10.1016/S0378-1127(97)00121-
7
Fan F, Henriksen CB, Porter J (2018) Relationship between
stoichiometry and ecosystem services: a case study of
organic farming systems. Ecol Indic 85:400–408. https://
doi.org/10.1016/j.ecolind.2017.10.063
Fernandez I, Mahieu N, Cadisch G (2003) Carbon isotopic
fractionation during decomposition of plant materials of
different quality. Glob Biogeochem Cycles 17:1075.
https://doi.org/10.1029/2001GB001834
Francey RJ, Allison CE, Etheridge DM, Trudinger CM, Enting
IG, Leuenberger M, Langenfelds RL, Michel E, Steele LP
(1999) A 1000-year high precision record of d
13
Cin
atmospheric CO
2
. Tellus 51B:170–193. https://doi.org/10.
1034/j.1600-0889.1999.t01-1-00005.x
Frouz J, Piz
ˇl V, Cienciala E, Kalc
ˇı
´k J (2009) Carbon storage in
post-mining forest soil, the role of tree biomass and soil
bioturbation. Biogeochemistry 94:111–121. https://doi.
org/10.1007/s10533-009-9313-0
Garten CT (2006) Relationships among forest soil C isotopic
composition, partitioning, and turnover times. Can J For
Res 36:2157–2167. https://doi.org/10.1139/x06-115
Garten CT, Cooper LW, Post M, Hanson PJ (2000) Climate
controls on forest soil C isotope ratios in the southern
Appalachian Mountains. Ecology 81(4):1108–1119
Gurmesa GA, Schmidt IK, Gundersen P, Vesterdal L (2013)
Soil carbon accumulation and nitrogen retention traits of
four tree species grown in common gardens. For Ecol
Manag 309:47–57. https://doi.org/10.1016/j.foreco.2013.
02.015
Handley LL, Raven JA (1992) The use of natural abundance of
nitrogen isotopes in plant physiology and ecology. Plant,
Cell Environ 15:965–985. https://doi.org/10.1111/j.1365-
3040.1992.tb01650.x
Harmon ME, Silver WL, Fasth B, Chen H, Burke IC, Parton WJ,
Hart SC, Currie WS, LIDET (2009) Long-term patterns of
mass loss during the decomposition of leaf and fine root
litter: an intersite comparison. Glob Change Biol
15:1320–1338. https://doi.org/10.1111/j.1365-2486.2008.
01837.x
Hobbie EA, Colpaert JV (2003) Nitrogen availability and col-
onization by mycorrhizal fungi correlate with nitrogen
isotope patterns in plants. New Phytol 157:115–126.
https://doi.org/10.1046/j.1469-8137.2003.00657.x
Hobbie EA, Ho
¨gberg P (2012) Nitrogen isotopes link mycor-
rhizal fungi and plants to nitrogen dynamics. New Phytol
196(2):367–382. https://doi.org/10.1111/j.1469-8137.
2012.04300.x
Hobbie EA, Ouimette AP (2009) Controls of nitrogen isotope
patterns in soil profiles. Biogeochemistry 95:355–371.
https://doi.org/10.1007/s10533-009-9328-6
Hobbie EA, Werner RA (2004) Intramolecular, compound-
specific, and bulk carbon isotope patterns in C3 and C4
plants: a review and synthesis. New Phytol 161:371–385.
https://doi.org/10.1046/j.1469-8137.2004.00970.x
Ho
¨gberg P (1997)
15
N natural abundance in soil-plant systems.
New Phytol 137:179–203. https://doi.org/10.1046/j.1469-
8137.1997.00808.x
Ho
¨gberg P, Ho
¨gbom L, Schinkel H, Ho
¨gberg M, Johannisson C,
Wallmark H (1996)
15
N abundance of surface soils, roots
and mycorrhizas in profiles of European forest soils.
Oecologia 108:207–214. https://doi.org/10.1007/
BF00334643
Jones DL, Nguyen C, Finlay RD (2009) Carbon flow in the
rhizosphere: carbon trading at the soil–root interface. Plant
Soil 321:5–33. https://doi.org/10.1007/s11104-009-9925-0
Kramer C, Trumbore S, Fro
¨berg M, Cisneros Dozal LM, Zhang
D, Xu X, Santos GM, Hanson PJ (2010) Recent (\4 year
old) leaf litter is not a major source of microbial carbon in a
temperate forest mineral soil. Soil Biol Biochem
42:1028–1037. https://doi.org/10.1016/j.soilbio.2010.02.
021
Kramer MG, Lajtha K, Aufdenkampe AK (2017) Depth trends
of soil organic matter C:N and 15 N natural abundance
controlled by association with minerals. Biogeochemistry
136:237–248. https://doi.org/10.1007/s10533-017-0378-x
Laganie
`re J, Pare
´D, Bradley RL (2010) How does a tree species
influence litter decomposition? Separating the relative
123
218 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

contribution of litter quality, litter mixing, and forest floor
conditions. Can J For Res 40:465–475. https://doi.org/10.
1139/X09-208
Lehmann J, Kleber M (2015) The contentious nature of soil
organic matter. Nature 528:60–68. https://doi.org/10.1038/
nature16069
Lerch TZ, Nunan N, Dignac MF, Chenu C, Mariotti A (2011)
Variations in microbial isotopic fractionation during soil
organic matter decomposition. Biogeochemistry
106(1):5–21. https://doi.org/10.1007/s10533-010-9432-7
Liese R, Lu
¨bbe T, Albers NW, Meier IC (2017) The mycorrhizal
type governs root exudation and nitrogen uptake of tem-
perate tree species. Tree Physiol 38:83–95. https://doi.org/
10.1093/treephys/tpx131
Lindahl BD, Tunlid A (2015) Ectomycorrhizal fungi—potential
organic matter decomposers, yet not saprotrophs. New
Phytol 205:1443–1447. https://doi.org/10.1111/nph.13201
Lorenz M, Thiele-Bruhn S (2019) Tree species affect soil
organic matter stocks and stoichiometry in interaction with
soil microbiota. Geoderma 353:35–46. https://doi.org/10.
1016/j.geoderma.2019.06.021
Manzoni S, Trofymow JA, Jackson RB, Porporato A (2010)
Stoichiometric controls on carbon, nitrogen, and phos-
phorus dynamics in decomposing litter. Ecol Monogr
80:89–106. https://doi.org/10.1890/09-0179.1
Marı
´n-Spiotta E, Gruley KE, Crawford J, Atkinson EE, Miesel
JR, Greene S, Cardona-Correa C, Spencer GM (2014)
Paradigm shifts in soil organic matter research affect
interpretations of aquatic carbon cycling: transcending
disciplinary and ecosystem boundaries. Biogeochemistry
117:279–297. https://doi.org/10.1007/s10533-013-9949-7
Marty C, Houle D, Gagnon C (2015) Effect of the relative
abundance of conifers versus hardwoods on soil d
13
C
enrichment with soil depth in Eastern Canadian forests.
Ecosystems 18:629–642. https://doi.org/10.1007/s10021-
015-9852-2
Marty C, Houle D, Courchesne F, Gagnon (2019) Soil C:N ratio
is the main driver of soil d15 N in cold and N-limited
eastern Canadian forests. CATENA 172:285–294. https://
doi.org/10.1016/j.catena.2018.08.029
Mueller KE, Eissenstat DM, Hobbie SE, Oleksyn J, Jagodzinski
AM, Reich PB, Chadwick OA, Chorover J (2012) Tree
species effects on coupled cycles of carbon, nitrogen, and
acidity in mineral soils at a common garden experiment.
Biogeochemistry 111:601–614. https://doi.org/10.1007/
s10533-011-9695-7
Mueller KE, Hobbie SE, Chorover J, Reich PB, Eisenhauer N,
Castellano MJ, Chadwick OA, Dobies T, Hale CM,
Jagodzin
´ski AM, Kałucka I, Kieliszewska-Rokicka B,
Modrzyn
´ski J, Ro_
zen A, Skorupski M, Sobczyk Ł, Sta-
sin
´ska M, Trocha LK, Weiner J, Wierzbicka A, Oleksyn J
(2015) Effects of litter traits, soil biota, and soil chemistry
on soil carbon stocks at a common garden with 14 tree
species. Biogeochemistry 123:313–327. https://doi.org/10.
1007/s10533-015-0083-6
Nadelhoffer KJ, Fry B (1988) Controls on natural nitrogen-15
and carbon-13 abundances in forest soil organic matter.
Soil Sci Soc Am J 52(6):1633–1640. https://doi.org/10.
2136/sssaj1988.03615995005200060024x
Nel JA, Craine JM, Cramer MD (2018) Correspondence
between d
13
C and d
15
N in soils suggests coordinated
fractionation processes for soil C and N. Plant Soil
423(1–2):257–271. https://doi.org/10.1007/s11104-017-
3500-x
Osono T, Takeda H, Azuma J (2008) Carbon isotope dynamics
during leaf litter decomposition with reference to lignin
fractions. Ecol Res 23:51–55. https://doi.org/10.1007/
s11284-007-0336-5
Pan Y, Birdsey RA, Fang J, Houghton R, Kauppi PE, Kurz WA,
Phillips OL, Shvidenko A, Lewis SL, Canadell JG, Ciais P,
Jackso RB, Pacala SW, McGuire AD, Piao AR, Rautiainen
A, Sitch S, Hayes (2011) A large and persistent carbon sink
in the world’s forests. Science 333:988. https://doi.org/10.
1126/science.1201609
Pardo LH, Hemond HF, Montoya JP, Pett-Ridge J (2007) Nat-
ural abundance
15
N in soil and litter across a nitrate-output
gradient in New Hampshire. For Ecol Manag 251:217–230.
https://doi.org/10.1016/j.foreco.2007.06.047
Pardo LH, Semaoune P, Schaberg PG, Eagar C, Sebilo M (2013)
Patterns in d
15
N in roots, stems, and leaves of sugar maple
and American beech seedlings, saplings, and mature trees.
Biogeochemistry 112:275–291. https://doi.org/10.1007/
s10533-012-9724-1
Paul EA (2016) The nature and dynamics of soil organic matter:
plant inputs, microbial transformations, and organic matter
stabilization. Soil Biol Biochem 98:109–126. https://doi.
org/10.1016/j.soilbio.2016.04.001
Poirier V, Roumet C, Munson AD (2018) The root of the matter:
linking root traits and soil organic matter stabilization
processes. Soil Biol Biochem 120:246–259. https://doi.org/
10.1016/j.soilbio.2018.02.016
Po
¨rtl K, Zechmeister-Boltenstern S, Wanek W, Ambus P, Ber-
ger TW (2007) Natural
15
N abundance of soil N pools and
N2O reflect the nitrogen dynamics of forest soils. Plant Soil
295:79–94. https://doi.org/10.1007/s11104-007-9264-y
Prescott CE (2010) Litter decomposition: what controls it and
how can we alter it to sequester more carbon in forest soils?
Biogeochemistry 101:133–149. https://doi.org/10.1007/
s10533-010-9439-0
R Core Team (2016) R: a language and environment for sta-
tistical computing. R Foundation for Statistical Comput-
ing, Vienna, Austria. https://www.R-project.org/
Reich PB, Oleksyn J, Modrzynski J, Mrozinski P, Hobbie SE,
Eissenstat DM, Chorover J, Chadwick OA, Hale CM,
Tjoelker (2005) Linking litter calcium, earthworms and
soil properties: a common garden test with 14 tree species.
Ecol Lett 8:811–818. https://doi.org/10.1111/j.1461-0248.
2005.00779.x
Rennenberg H, Dannenmann M, Gessler A, Kreuzwieser J,
Simon J, Papen H (2009) Nitrogen balance in forest soils
nutritional limitation of plants under climate change
stresses. Plant Biol 11:24–33. https://doi.org/10.1111/j.
1438-8677.2009.00241.x
Schrumpf M, Kaiser K, Guggenberger G, Persson T, Ko
¨gel-
Knabner I, Schulze ED (2013) Storage and stability of
organic carbon in soils as related to depth, occlusion within
aggregates, and attachment to minerals. Biogeosciences
10:1675–1691. https://doi.org/10.5194/bg-10-1675-2013
Schwartz E, Blazewicz S, Doucett R, Hungate BA, Hart SC,
Dijkstra P (2007) Natural abundance d
15
N and d
13
Cof
DNA extracted from soil. Soil Biol Biochem
123
Biogeochemistry (2020) 151:203–220 219
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

39:3101–3107. https://doi.org/10.1016/j.soilbio.2007.07.
004
Silfer JA, Engel MH, Macko SA (1992) Kinetic fractionation of
stable carbon and nitrogen isotopes during peptide bond
hydrolysis: experimental evidence and geochemical
implications. Chem Geol: Isotope Geosci Sect
101(3–4):211–221. https://doi.org/10.1016/0009-
2541(92)90003-N
Stevenson FJ (1994) Humus chemistry—genesis, composition,
reactions, 2nd edn. Wiley, New York
Sua
´rez-Abelenda M, Ahmad R, Camps-Arbestain M, Herath
SHMSK (2015) Changes in the chemical composition of
soil organic matter over time in the presence and absence of
living roots: a pyrolysis GC/MS study. Plant Soil
391:161–177. https://doi.org/10.1007/s11104-015-2423-7
Templer PH, Arthur MA, Lovett GM, Weathers KC (2007)
Plant and soil natural abundance d
15
N: indicators of rela-
tive rates of nitrogen cycling in temperate forest ecosys-
tems. Oecologia 153:399–406. https://doi.org/10.1007/
s00442-007-0746-7
Trumbore SE (2009) Radiocarbon and soil carbon dynamics.
Annu Rev Earth Planet Sci 37:47–66. https://doi.org/10.
1146/annurev.earth.36.031207.124300
Ukonmaanaho L, Pitman R, Bastrup-Birk A, Breda N, Rautio P
(2016) Part XIII: Sampling and Analysis of Litterfall. In:
UNECE ICP Forests Programme Co-ordinating Centre
(ed) Manual on methods and criteria for harmonized
sampling, assessment, monitoring and analysis of the
effects of air pollution on forests. Thu
¨nen Institute for
Forests Ecosystems, Eberswalde, Germany, 14 p. ?An-
nex. http://www.icp-forests.org/manual.htm
Vallano DM, Sparks JP (2013) Foliar d
15
N is affected by foliar
nitrogen uptake, soil nitrogen, and mycorrhizae along a
nitrogen deposition gradient. Oecologia 172:47–58. https://
doi.org/10.1007/s00442-012-2489-3
Vesterdal L, Schmidt IK, Callesen I, Nilsson LO, Gundersen P
(2008) Carbon and nitrogen in forest floor and mineral soil
under six common European tree species. For Ecol Manag
225:35–48. https://doi.org/10.1016/j.foreco.2007.08.015
Vesterdal L, Elberling B, Christiansen JR, Callesen I, Schmidt
IK (2012) Soil respiration and rates of soil carbon turnover
differ among six common European tree species. For Ecol
Manag 264:185–196. https://doi.org/10.1016/j.foreco.
2011.10.009
Vesterdal L, Clarke N, Sigurdsson BD, Gundersen P (2013) Do
tree species influence soil carbon stocks in temperate and
boreal forests? For Ecol Manag 309:4–18. https://doi.org/
10.1016/j.foreco.2013.01.017
von Lu
¨tzow M, Ko
¨gel-Knabner I, Ekschmitt K, Matzner E,
Guggenberger G, Marschner B, Flessa H (2007) Stabi-
lization of organic matter in temperate soils: mechanisms
and their relevance under different soil conditions—a
review. Eur J Soil Sci 57:426–445. https://doi.org/10.1111/
j.1365-2389.2006.00809.x
Wallander H, Mo
¨rth CM, Giesler R (2009) Increasing abun-
dance of soil fungi is a driver for
15
N enrichment in soil
profiles along a chronosequence undergoing isostatic
rebound in northern Sweden. Oecologia 160:87–96. https://
doi.org/10.1007/s00442-008-1270-0
Wang B, Qiu YL (2006) Phylogenetic distribution and evolution
of mycorrhizas in land plants. Mycorrhiza 16:299–363.
https://doi.org/10.1007/s00572-005-0033-6
Wang G, Jia Y, Li W (2015) Effects of environmental and biotic
factors on carbon isotopic fractionation during decompo-
sition of soil organic matter. Sci Rep 5:11043. https://doi.
org/10.1038/srep11043
Wang C, Houlton BZ, Liu D, Hou J, Cheng W, Bai E (2018)
Stable isotopic constraints on global soil organic carbon
turnover. Biogeosciences 15:987–995. https://doi.org/10.
5194/bg-15-987-2018
Werth M, Kuzyakov Y (2010)
13
C fractionation at the root–
microorganisms–soil interface: a review and outlook for
partitioning studies. Soil Biol Biochem 42:1372–1384.
https://doi.org/10.1016/j.soilbio.2010.04.009
Wilske B, Eccard JA, Zistl-Schlingmann M, Hohmann M,
Methler A, Herde A, Liesenjohann T, Dannenmann M,
Butterbach-Bahl K, Breuer L (2015) Effects of short term
bioturbation by common voles on biogeochemical soil
variables. PLoS ONE 10(5):e0126011. https://doi.org/10.
1371/journal.pone.0126011
Wynn JG, Harden JW, Fries TL (2006) Stable carbon isotope
depth profiles and soil organic carbon dynamics in the
lower Mississippi Basin. Geoderma 131:89–109. https://
doi.org/10.1016/j.geoderma.2005.03.005
Yuan ZY, Chen HYH (2010) Fine root biomass, production,
turnover rates, and nutrient contents in boreal forest
ecosystems in relation to species, climate, fertility, and
stand age: literature review and meta-analyses. Crit Rev
Plant Sci 29:204–221. https://doi.org/10.1080/07352689.
2010.483579
Zanella A, Ponge J-F, Jabiol B, Sartori G, Kolb E, Le Bayon
R-C, Gobat J-M, Aubert M, De Waal R, Van Delft B,
Vacca A, Serra G, Chersich S, Andreetta A, Ko
˜lli R, Brun
JJ, Cool N, Englisch M, Hager H, Katzensteiner K, Bre
ˆthes
A, De Nicola C, Testi A, Bernier N, Graefe U, Wolf U,
Juilleret J, Garlato A, Obber S, Galvan P, Zampedri R,
Frizzera L, Tomasi M, Banas D, Bureau F, Tatti D, Salmon
S, Menardi R, Fontanella F, Carraro V, Pizzeghello D,
Concheri G, Squartini A, Cattaneo D, Scattolin L, Nardi S,
Nicolini G, Viola F (2018) Humusica 1, article 5: terrestrial
humus systems and forms—keys of classification of humus
systems and forms. Appl Soil Ecol 122:75–86. https://doi.
org/10.1016/j.apsoil.2017.06.012
Zechmeister-Boltenstern S, Keiblinger KM, Mooshammer M,
Pen
˜uelas J, Richter A, Sardans J, Wanek W (2015) The
application of ecological stoichiometry to plant–micro-
bial–soil organic matter transformations. Ecol Monogr
85:133–155. https://doi.org/10.1890/14-0777.1
Zeller B, Legout A, Bienaime
´S, Gratia B, Santenoise P, Bon-
naud P, Ranger J (2019) Douglas fir stimulates nitrification
in French forest soils. Sci Rep 9:10687. https://doi.org/10.
1038/s41598-019-47042-6
Publisher’s Note Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations.
123
220 Biogeochemistry (2020) 151:203–220
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”),
for small-scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are
maintained. By accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use
(“Terms”). For these purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or
a personal subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or
a personal subscription (to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the
Creative Commons license used will apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data
internally within ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking,
analysis and reporting. We will not otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of
companies unless we have your permission as detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that
Users may not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to
circumvent access control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil
liability, or is otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by
Springer Nature in writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer
Nature journal content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates
revenue, royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain.
Springer Nature journal content cannot be used for inter-library loans and librarians may not upload Springer Nature journal
content on a large scale into their, or any other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any
information or content on this website and may remove it or features or functionality at our sole discretion, at any time with or
without notice. Springer Nature may revoke this licence to you at any time and remove access to any copies of the Springer Nature
journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express
or implied with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or
warranties imposed by law, including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be
licensed from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other
manner not expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com