Supporting Information (SI)
Ammonium first: natural mosses prefer atmospheric
ammonium but vary utilization of dissolved organic nitrogen
depending on habitat and nitrogen deposition
Xue-Yan Liu1, 2, Keisuke Koba2*, Akiko Makabe2, Xiao-Dong Li1, Muneoki Yoh2,
Cong-Qiang Liu1
1 State
Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese
Academy of Sciences, Guiyang, 550002, China; 2 Faculty of Agriculture, Tokyo University of
Agriculture and Technology, Fuchu 1838509, Japan
*
Author for correspondence: Keisuke Koba
E-mail: keikoba@cc.tuat.ac.jp; Tel and fax: +81-42-367-5951
Index:
Supporting Information
Fig. S1 Map showing the location of Guiyang area and sampling sites.
Fig. S2 Contributions of soil-RDN to moss bulk N at Guiyang area.
Fig. S3 Contributions of NH4+ and DON to moss bulk N at Guiyang area.
Fig. S4 Soil N availability vs soil N contribution in moss N at Guiyang area.
Table S1 Level and δ15N of dissolved N in wet deposition at Guiyang, SW China.
Table S2 Bulk N and δ15N in natural mosses at Guiyang area.
Notes S1 Additional methodological details on study area, precipitation collection,
isotopic analyses of soil N and wet-deposited N.
Notes S2 Additional results of characteristics of soils under mosses.
Notes S3 Additional results of isotopic mass-balance calculations based on RDN.
S1
Figure S1
Fig. S1. Map showing the location of Guiyang area and sampling sites of natural
mosses (after Liu et al., 2012c). R, S and U represent rural, suburban and urban sites,
respectively.
S2
Figure S2
80
fsoil-RDN in moss Nbulk (%)
70
60
ES
TO
TF
Average
b
50
b
b
40
b
b
ab
a
30
a
a
20
b
b
a
10
0
Urban
Suburban
Rural
Fig. S2. Proportional contributions of soil RDN (fsoil-RDN) to moss Nbulk estimated by
the δ15N mass balance calculation (Different letters above the bars showed statistical
difference between means ± SD at the significance level of P < 0.05). ES for epilithic
mosses with soils, TO for terricolous mosses in open fields and TF for mosses on
floors of pine forests.
S3
Figure S3
Urban
Suburban
Rural
EB
ES
TO
TF
EB
ES
TO
TF
EB
ES
TO
atm-NH4+
TF
Urban
Suburban
Rural
EB
ES
TO
TF
EB
ES
TO
TF
EB
ES
TO
atm-DON
TF
ES
Urban TO
TF
ES
Suburban TO
TF
Rural
ES
TO
soil-NH4+
TF
ES
Urban TO
TF
ES
Suburban TO
TF
Rural
ES
TO
soil-DON
TF
0
10
20
30
40
50
60
70
80
90
100
Proportional contribution (%)
Fig. S3. Contributions of NH4+ (atm-NH4+ and soil-NH4+) and DON (atm-DON and
soil-DON) to bulk N assimilation in natural mosses at Guiyang area. Calculations
were conducted on individual samples by the two-source mixing model (Eqn. 1; for
EB) and the ‘IsoSource’ model (Eqn. 3; for ES, TO and TF). The output of
‘IsoSource’ model is a wide range for each N source in each replicate sample. The
reported ranges and mean values were original data from the model, not mean values
of each replicate. The solid and dash lines within the boxes mark the median and the
mean, the boundary of the box indicates the 25th and 75th percentiles, and the dots
above and below the whisker caps indicate the 5th and 95th percentiles, respectively.
EB represents for epilithic mosses on bare rocks, ES for epilithic mosses with soils,
TO for terricolous mosses in open fields and TF for mosses on floors of pine forests.
S4
Figure S4
2
Proportional contribution (%) R = 0.06, P = -0.22
-1
2
Real contribution (mg-N g , dw) R = 0.01, P = 0.63
2
Proportional contribution (%) R = 0.00, P = -0.78
-1
2
Real contribution (mg-N g , dw) R = 0.00, P = 0.80
Contribution of soil-DON in N moss
Contribution of soil-NH 4+ in Nmoss
30
a
20
10
0
0
10
Soil
20
NH4+
30
40
50
60
60
b
50
40
30
20
10
0
0
70
20
40
60
80
Soil DON availability (mg-N kg-1, dw)
-1
availability (mg-N kg , dw)
Fig. S4. The relationships between soil N availability (a: soil-NH4+; b: soil-DON) and
corresponding proportional or real contribution of soil N in bulk N of mosses at
Guiyang area.
Table S1. Level and δ15N signatures of dissolved N in wet deposition at Guiyang, SW
China. Values in () show the data range (n = 44, from Dec-2008 to Sep-2009).
N form
Deposition level
(kg-N ha-1 yr-1)
Proportion (%)
in TDN
in RDN
TDN
30.7
-
-
-6.3±5.5 (-18.0−7.4)
Liu et al. 2012
RDN
25.4
83
-
-7.5±7.2 (-22.9−9.7)
Liu et al. 2012
+
15.6
9.8
5.3
51
32
17
62
38
-
-14.8±3.0 (-18.8−-10.4)
6.8±15.7 (-6.8−40.7)*
1.9±3.0 (-8.3−2.9)
Xiao et al. 2012
Liu et al. 2012; This study
Liu et al. 2012
NH4
DON
NO3-
δ15N / ‰
References
* calculated based on the monthly-averaged data (n = 10) for concentrations and isotopic signatures
from Liu et al (2012c) and Xiao et al (2012), thus this data was calculated in the different way as we
did for soil DON (based on each replicate soil sample; See Methods in the main text).
Table S2. Bulk N and δ15N in natural mosses at Guiyang area (Data were cited from
Liu et al., 2012c). Reported are means ± SD.
Site
n
Moss N (mg g-1, dw)
Moss δ15N / ‰
Urban
6
23.6 ± 1.8
-7.9 ± 3.3
EB
Suburban
6
17.4 ± 1.9
-4.0 ± 0.3
Rural
5
16.8 ± 3.0
-3.9 ± 0.7
Urban
3
25.3 ± 1.1
-7.7 ± 3.2
ES
Suburban
3
19.8 ± 2.1
-2.8 ± 0.3
Rural
3
19.9 ± 1.4
-3.2 ± 0.3
Urban
3
20.5 ± 4.0
-5.3 ± 1.5
TO
Suburban
3
23.9 ± 2.3
-2.0 ± 0.3
Rural
3
20.6 ± 2.6
-2.4 ± 0.3
Urban
3
18.6 ± 2.3
-5.6 ± 0.5
TF
Suburban
3
15.6 ± 2.2
-4.0 ± 0.6
Rural
3
17.8 ± 2.0
-2.4 ± 1.0
EB: mosses on bare rock; ES: mosses with soil on rock; TO: terricolous mosses in open fields; TF:
terricolous mosses in pine forests.
Habitat
S5
Notes S1 Additional methodological details on study area,
precipitation collection, analyses of concentrations and stable
isotopes of soil N and wet-deposited N.
Study area
Guiyang area has a typical subtropical monsoon climate, and most of the landforms
are in the altitude of 1000 − 1500 m (average 1250 m). The mean annual temperature,
relative humidity and precipitation is 15.3 ºC, 86% and 1174 mm, respectively. A
decrease of N levels (31 kg-N ha-1 yr-1 in the urban to 11 kg-N ha-1 yr-1 in rural areas)
and the dominance of RDN have been observed in atmospheric depositions across the
urban–rural transect (Liu et al., 2012c).
Precipitation collection
Bulk precipitation was collected in an urban site (near U1, Fig. S1) from Dec. 2008 to
Sept. 2009. The sampling site was the same as that described in Xiao & Liu (2002,
2004). Details of sample collection have been reported in Xiao et al. (2012). Briefly,
precipitation was continuously sampled using 1.5L container (light rain events) and
5L container (heavy rain events), i.e., from the beginning to the end of each rain event.
As a result, most rain samples (1235 in total) were not on a daily basis, but divided
during the course of single rain event. Many of event samples had extremely high or
low ratios of RDN to NO3-N, some of which contained no nitrate or too low to be
measured for isotopes of major N species. Therefore, we randomly chose 44
daily-based rain samples (n = 3−6 for each month) roughly distributed from the
beginning to the end of each month for isotopic analyses of explicit N components.
Samples were frozen (at -20°C) and shipped to Japan.
Analyses of soil N concentrations and stable isotopes
The NH4+ concentration in soil extracts was determined by the indophenol blue
method followed by colorimetry, the NO3- concentration was colorimetrically
determined by an autoanalyzer (TRAACS 800; Bran Luebbe GmbH, Tokyo, Japan)
(Koba et al., 2010a,b). The TDN in 10 ml of soil extract was digested to NO3- using
alkaline persulfate digestion (Koba et al., 2010a,b; Koba et al., 2012) and measured as
NO3- on the autoanalyzer. The RDN was calculated as the difference between TDN
and NO3-. The DON was calculated as the difference between RDN and NH4+ or TDN
and DIN. The sieved fresh soils were immediately incubated for 28 days at 25 °C for
measuring net rates of N mineralization and nitrification. At the end of the incubation,
DIN was measured for incubated soils using the same methods mentioned above. The
net N mineralization was calculated as differences between initial and incubated DIN,
and nitrification as difference between initial and incubated NO3-. Percent nitrification
was defined as the percentage of net nitrification rate to net N mineralization rate.
Mosses and soils were analyzed for Nbulk and δ15Nbulk using an elemental analyzer
(EA1112; Thermo Fisher Scientific K.K., Yokohama, Japan) coupled with an isotope
ratio mass spectrometer (Delta-XP; Thermo Fisher Scientific K.K.). Calibrated
DL-alanine (δ15N = -1.7‰), glycine (δ15N = 10.0‰), and histidine (δ15N = -8.0‰)
against IAEA-N-1 (ammonium sulfate) were used as the internal standards. The
analytical precision for δ15N was better than 0.2‰. Stable isotopes of NO3- in soil
extracts were measured using the denitrifier method (Sigman et al. 2001; Casciotti et
S6
al. 2002; Koba et al., 2010) on the Delta XP isotope ratio mass spectrometer coupled
with a gas chromatograph (HP6890; Hewlett Packard Co., Palo Alto, CA, USA)
equipped with a Poraplot column (25m × 0.32 mm), Precon (Thermo Fisher Scientific
K.K.), and GC interface III (Thermo Fisher Scientific K.K.). The denitrifying bacteria
(Pseudomonas aureofaciens; ATCC 13985) was used to convert 20 nmoles of NO3into gaseous N2O in 20 − 60 ml vials prior to isotopic analyses. We ran several
standards (USGS 32, 34, 35 and IAEA NO3-) dissolved in the same KCl solution for
soil extraction. The average standard deviations for replicate analysis of an individual
sample were ± 0.2‰ (SD) for δ15N in NO3- and ± 0.5‰ (SD) for δ18O. The δ15N of
TDN were determined by analyzing the persulfate-digested samples using the same
method as that used for NO3-. We ran several standards (alanine, glycine and histidine,
with known δ15N values used for EA-IRMS analysis above) dissolved in the KCl
solution for calibrating the δ15N of TDN. We also analyzed several TDN samples for
the KCl blank (1 − 5 ml of digested KCl solution) to correct for the effect of the N
blank from the KCl solution. The average standard deviation for replicate analyses of
δ15N-TDN in an individual sample was ± 0.3‰ (SD).
The diffusion method was used to recover NH4+ from the soil extract. 5 ml of soil
extract was pipetted into a 10 ml glass vial (No. 8; Maruemu Corp. Co. Ltd., Osaka,
Japan) immediately after the extraction in the laboratory at the Institute of
Geochemistry, China. A glass fiber filter (GF/D, 1 cm diameter, treated at 450°C for 4
h; Whatman Int. Ltd.) acidified with 20 μl of 2 M H2SO4 and wrapped with Teflon
tape was added to the vial. After the addition of 0.02 g of MgO (treated at 450°C for 4
h), the vial was quickly closed with an aluminum crimp cap and a butyl rubber stopper
(anaerobic stopper; Kandagomukagaku Corp., Tokyo, Japan). We used a constant
volume (5 ml) for samples and standards to correct the δ15N affected by the N blank
from KCl and the reagents in this diffusion procedure (from MgO, glass fiber filter,
diluted H2SO4 and atmospheric ammonia). Several standards and lab working
standards (USGS25, 26 and IAEA N-2 dissolved in 5 ml of KCl solution) were
diffused as samples to obtain the calibration curve. Several blank samples (5 ml of
KCl solution) were also run in parallel. The vials were incubated at 40 °C for 14 days
using an oven-shaker (FMS-1000 and MMS-3010; Tokyo Rikakikai Co. Ltd., Tokyo,
Japan) with a horizontal shaking speed of 180 rpm. The concentrated NH4+ on the
glass fiber filter was digested to NO3- using persulfate oxidation, then the δ15N of
NH4+ was measured as the NO3- using the denitrifier method. The average standard
deviation for triplicate analyses of an individual sample for δ15N of NH4+ was ± 0.5‰
after the blank correction.
Analyses of wet-deposited N concentrations and stable isotopes
Chemical analyses of NH4+, NO3-, TDN concentrations in precipitation were the same
as that described in Li et al (2012), which was conducted in the same laboratory. The
RDN was calculated as the difference between TDN and NO3-. The DON was
calculated as the difference between TDN and DIN. Isotopic analyses of rain NO3and TDN were the same as those of soil extracts described above. The δ15N data of
NH4+ was cited from Xiao et al (2012). The δ15N of RDN and DON were calculated
using the following mass and isotopic balance equations:
δ15NRDN = {δ15NTDN × [TDN] – δ15NNO3 × [NO3--N]} / [RDN] (‰).
δ15NDON = {δ15NTDN × [TDN] – δ15NNO3- × [NO3−] – δ15NNH4+ × [NH4+]} / [DON]
(‰).
S7
Notes S2 Additional results of characteristics of soils under
mosses.
The pH of substrate soils did not differ between epilithic (6.5) and terricolous mosses
(6.2) in open fields, but moss soils in pine forests had a significantly lower pH (5.6),
with the lowest value in the urban area (Table S1). The C of moss soils (3.2 – 13.1%)
showed the highest and lowest concentrations in organic soil layers of epilithic
mosses and soils in pine forests, respectively. The bulk N of moss soils (0.3 – 0.7%)
decreased along the urban-rural gradient except of that in pine forests which had the
lowest N level (Table S1).
Net nitrification and mineralization rates of moss substrate soils had a range of
0.1 – 1.2 mg-N kg-1 day-1 and 0.3 – 5.4 mg-N kg-1 day-1, respectively, showing a
decrease from the urban to rural sites (Table S1). Soils of epilthic mosses showed
higher net N rates than those of terricolous mosses. Soils in pine forests had the
lowest net nitrification rates (0.1 – 0.3 mg-N kg-1 day-1) and magnitudes of
nitrification (5 – 10%) (Table S1). A positive correlation was observed between soil
net N rates and pH values (P < 0.01) (Table S2).
Notes S3 Additional results of calculations of the deposited RDN
and substrate RDN contributions to moss bulk N
The contributions of soil RDN (fsoil-RDN) and wet-deposited RDN (fatm-RDN) to N in
mosses on soils (Nmoss-soil) could also be estimated by the two-source mixing model:
δ15Nmoss-soil = fsoil-RDN × δ15Nsoil-RDN + fatm-RDN × δ15Natm-RDN.
(Eqn. S1)
Since fsoil-RDN + fatm-RDN = 1 and δ15Natm-RDN = -7.5‰ (Table S1), the Eqn. S1 can be
simplified to:
fsoil-RDN = [(δ15Nmoss + 7.5) / (δ15Nsoil-RDN + 7.5)] × 100,
(Eqn. S2)
The results showed that the fsoil-RDN averaged 30 ± 14% (Fig. S2). Moreover, the
fsoil-RDN in mosses showed a pattern being opposite with that of N deposition level.
The lowest fsoil-RDN (10 ± 15%) was observed for urban epilithic mosses where N
deposition was high although urban substrates also had high N availability. In contrast,
the highest fsoil-RDN (46 ± 10%) was found in rural forest mosses with low N
deposition and soil N availability.
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