DOI: 10.5061/dryad.h44j0zpq1
title: Isotopic evidence for increased carbon and nitrogen exchanges between peatland plants and their symbiotic microbes with rising atmospheric CO2 concentrations since 15000 cal. yr BP

description: Whether nitrogen (N) availability will limit plant growth and removal of
 atmospheric CO2 this century is controversial. Studies have suggested that
 N could progressively limit plant growth, as trees and soils accumulate N
 in slowly cycling biomass pools in response to increases in carbon
 sequestration. However, a question remains over the longer-term (decadal
 to century) feedbacks between climate, CO2 and plant N uptake. The
 symbiosis between plants and microbes can help plants with mycorrhizal N
 uptake or biological N2 fixation – the pathway through which N can be
 rapidly brought into ecosystems and thereby partially or completely
 alleviate N limitation on plant productivity. Here we present results for
 plant N isotope composition (δ15N) in a peat core that dates to 15000 cal.
 yr BP to ascertain ecosystem-level N cycling responses to rising
 atmospheric CO2 concentrations in the past. We found that an increase in
 atmospheric CO2 concentration happened with a decrease in δ15N values of
 both Sphagnum moss and Ericaceae over this time period
 when constrained for climatic factors. A modern experiment demonstrated
 that δ15N of Sphagnum mosses decreased with increasing
 N2 fixation rates. These findings suggested that
 N2 fixation in Sphagnum moss by symbiosis with cyanobacteria and
 N uptake in Ericaceae by symbiosis with mycorrhizal fungi both likely
 increased with rising atmospheric CO2 concentrations, highlighting a
 longer-term feedback mechanism whereby N constraints on terrestrial carbon
 storage can be overcome. 
description: <strong>1. Study sites and sampling
 monoliths</strong> The peat core (42.227° N,
 126.525° E) was collected in Hani, a valley peatland situated in the
 middle of the Longgang Mountain Chain which is part of the western flank
 of the Changbai Mountains, Northeast China. The present surface vegetation
 community mainly comprises of <em>Betula</em>
 <em>ovalifolia</em>, <em>Ledum</em>
 <em>palustre</em>, <em>Phragmites</em>
 <em>australis</em>, <em>Vaccinium</em>
 <em>uliginosum</em>, <em>Carex</em>
 <em>limosa</em>, <em>S</em>.
 <em>palustre</em>, <em>S</em>.
 <em>magellanicum</em>, <em>S</em>.
 <em>cuspidatum</em>, <em>S</em>.
 <em>subsecundum</em>, <em>S</em>.
 <em>imbricatum</em>, <em>Polytrichum</em>
 <em>strictum</em> and <em>Aulacomnium</em>
 <em>palustre</em>. The mean annual temperature and
 precipitation range from 0.8 to 5.4 ℃ and from 490 to 1300 mm,
 respectively from local instrumental records (Jingyu County) during
 1955–2019 AD. The length of this core was 8.77 m. The peat core was
 described and cut with stainless steel at 1 cm intervals in the field. All
 of the samples were packed in plastic bags and stored at 4 ℃ in a
 refrigerator for further analyses. <strong>2.
 Radiocarbon dating</strong> The chronology of the
 peat core was based on the <sup>14</sup>C accelerator mass
 spectrometer (AMS) analyses of plant residual stems and leaves subjecting
 to an acid–alkali–acid treatment (Table S1). The graphite samples were
 finished in our <sup>14</sup>C dating preparation lab and then
 sent to the NTUAMS laboratory at the National Taiwan University (NTU) for
 AMS measurement with a HVE 1.0 MV Tandetron Model 4110 BO-Accelerator Mass
 Spectrometer (AMS). The measured <sup>14</sup>C ages were
 converted to calibrated calendar ages (a BP = years before 1950 AD) with
 the IntCal13 calibration curve (Reimer et al., 2013) using the CALIB 8.10
 program and expressed as cal. yr BP with 2σ ranges. The calendar age was
 calibrated using the Bacon age-depth model (Figure S1).
 <strong>3. Plant macrofossil preparation
 procedures</strong> Sub-samples (1 cm3) for plant
 macrofossils were analyzed using standard method (Mauquoy et al., 2010).
 Plant residues were washed with deionized water after alkaline solution
 treatment and then examined under a stereomicroscope at 10–20
 magnification and macrofossils were identified. Moss leaves and small
 seeds were examined at high magnifications (×100–400). Five plant groups:
 Ericaceae, Carex, <em>Equisetum</em>
 <em>fluviatile</em> (<em>E</em>.
 <em>fluviatile</em>), Sphagnum (<em>S</em>.
 <em>palustre</em>) and
 <em>Drepanocladus</em> (washed only with deionized water) were
 picked for further isotope analyses because of their high abundance
 (&gt; 10 mg for each sample) along the whole core and their residues
 were stored in a 5 mL centrifuge tube in a 4 ℃ refrigerator. Tilia 2.0.41
 (Grimm, 1992) was used to calculate plant macrofossil percentages and to
 plot the diagram. <strong>4. Stable nitrogen
 isotope analyses</strong> Each residue sample of
 the five selected groups was dried at 50 ℃ for 48 h, homogenized in a ball
 mill, and analyzed for N concentration and isotopic abundance in an
 elemental analyzer coupled online with an isotope ratio mass spectrometer
 (DELTA V Advantage; ThermoFisher, Germany). N isotopic
 abundance was expressed as δ15N and was calculated as follows: δ (‰) =
 [(R<sub>sample</sub>/R<sub>standard</sub>)-1)],
 where R is the <sup>15</sup>N/1<sup>4</sup>N ratio
 of the sample and the correspondent standard, air
 N<sub>2</sub>. To check the system stability, standard
 materials with known δ<sup>15</sup>N values were analyzed
 between every ten samples. Standard deviations of laboratory standards for
 δ<sup>15</sup>N were less than 0.15 ‰.
 <strong>5. Analysis of bulk peat and calculation of
 nitrogen accumulation rate (NAR)</strong> Each
 peat sample was weighed before and after drying at 105 ℃ until constant
 weight was reached. Dry bulk density (DBD) was calculated from the dry
 weight and sample volume. The dried samples were milled and homogenized
 for C, N content and C/N ratio analysis. Nitrogen accumulation rate (NAR)
 was calculated by the following equation based on raw data including peat
 accumulation rates (PAR), DBD and N contents. NAR = PAR * DBD * N (%) *
 100. Peat accumulation rates (PAR) (cm yr<sup>-1</sup>) for
 each profile were calculated for peat layers between the dated depths of
 the peat profile (Tolonen and Turunen, 1996).
 <strong>6. CO2 enrichment experiment of living Sphagnum and
 calculations of N<sub>2</sub> fixation
 rates</strong> In early September 2020, the
 dominant Sphagnum species (<em>S</em>.
 <em>fallax</em>, <em>S</em>.
 <em>magellanicum</em> and <em>S</em>.
 <em>fuscum</em>) were collected from both hollows and hummocks
 in Dongfanghong peatland, Northeast China (42.185°N, 128.312°E). This
 experiment had two periods: the treatment period (one month) and the assay
 period (48 h). First, we incubated the <em>Sphagnum</em> in
 lab for a month under two treatments (ambient
 CO<sub>2</sub> and elevated CO<sub>2</sub>
 with 600 ppm) with seven replicates. Plants were incubated under
 prevailing light conditions (7200 lx, 400–700 nm, day length of 16 h) and
 in the dark (0 lx, night length of 8h) and at 25 ℃ for daytime and 20 ℃
 for night in incubators. Distilled water was added into each sample every
 two days to prevent overdrying and maintain the original water
 level. After treatment period,
 <em>Sphagnum</em> mosses were incubated for assay of
 N<sub>2</sub>-fixation rates. Samples were incubated in 120 mL
 glass vials and 10 mL of water was added into the vials to keep the
 samples moist. A volume of 20 mL of air was removed, and 20 mL of 15N2
 tracer gas [98% (vol/vol)] were injected into the vials to reach headspace
 enrichment levels of 16% (vol/vol) for
 <sub>15</sub>N<sub>2</sub>. Incubations were
 terminated after 48 h by opening the vials, emptying water, and freezing
 them at -20 ℃. Then the samples were dried at 50 ℃ to a constant mass and
 analyzed for their bulk δ<sup>15</sup>N values and N contents
 using a stable isotope ratio mass spectrometer (DELTA V Advantage;
 ThermoFisher, Germany). Fe and Mo (below detection limit) contents in the
 tissue of the incubated <em>Sphagnum</em> samples were
 determined using sector field ICP-MS (7500C; ThermoFisher, Germany)
 employing well established analytical procedures (Kempter et al., 2017).
 Powdered samples were dissolved in a microwave autoclave (CEM, Mars6)
 using 3 mL high purity HNO<sub>3</sub> and 0.1 mL
 HBF<sub>4</sub>. The certified plant material (BW0000-2016)
 was used for quality control. 
 N<sub>2</sub> fixation rate was calculated according
 to Leppänen et al. (2013): N uptake (nmol
 g<sup>-1</sup> h<sup>-1</sup>) = (1/100) *
 (%N/100) * [atom%<sub>sample</sub> -
 atom%control/MW(N<sub>2</sub>)] *
 [(100/atom%<sub>headspace</sub>)/48] *
 10<sup>9</sup> (Eqn. 1); where % N is the N percent of the
 dried sample, and MW(N2) is molecular weight for
 N2 (28.0134462); atom%headspace =
 f<sub>N2</sub> * (atom%<sub>N2</sub>) -
 atom%<sub>15N2</sub>) + atom%<sub>15N2</sub>(Eqn.
 2); where f<sub>N2</sub> is the percentage for N2 in air or
 water, atom%<sub>N2</sub> = 0.36630,
 and atom%<sub>15N2</sub> = 98;
 f<sub>N2</sub> =
 V<sub>N2</sub>/(V<sub>N2</sub> +
 V<sub>15N2</sub>)  (Eqn. 3); where V is the volume of the
 corresponding gas. <strong>7. Data
 of atmospheric CO<sub>2</sub> concentrations and other
 hydroclimate factors </strong> Continuous records
 of the atmospheric CO<sub>2</sub> concentrations were from
 published ice core records and recent measurements on firn air and
 atmospheric samples spanning the interval from the penultimate glacial
 maximum (～156 kyr BP) to the beginning of the year 2016 AD (Köhler et al.,
 2017). The newest published age scales were used for the ice core data and
 atmospheric CO<sub>2</sub> concentrations represented an
 integrated global signal in this paper. We used raw data instead of
 splined-smoothed calculations. Meteorological data of mean annual
 temperature (MAT) had two sources depending on time. From 2010–1950 AD,
 the hydroclimate factors were from local instrumental records (Jingyu
 County). Before 1950 AD, the hydroclimate data were from previous
 publications: MAT was based on air temperature derived from brGDGT
 distributions with calibration in the same peatland as our sampling site
 of the peat core (Zheng et al., 2017). DWT was reconstructed by a transfer
 function (WATOL-inv) using plant macrofossil of the peat core (Yang et
 al., 2022). All the climatic factors were matched to plant
 δ<sup>15</sup>N data within plus and minus five
 years.  <strong>8. Statistical
 analyses</strong> Normality assumption was tested
 with Shapiro-Wilk´s test and homogeneity of variances was assessed using
 Bartlett´s test. Treatment effects were tested using one-way analysis of
 variance (ANOVA) and a Tukey HSD post-hoc test. The level of significance
 (α) was set at 0.05. In addition, bivariate relationships between plant
 δ<sup>15</sup>N and climatic factors were estimated using
 zero-order correlations (same as Pearson’s correlations) and partial
 correlations by controlling for a single edaphic variable (i.e. DWT, MAT,
 TN, CO2). The detailed significance level of the partial correlation
 matrix was shown in Table S2. All statistical analyses were conducted
 using SPSS 19.0 (SPSS Inc., Chicago, IL, USA).