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Environmental Processes

Magnitude and pathways of increased nitrous oxide emissions from uplands following permafrost thaw Guibiao Yang, Yunfeng Peng, Maija Marushchak, Yongliang Chen, Guanqin Wang, Fei Li, Dianye Zhang, Jun Wang, Jianchun Yu, Li Liu, Shuqi Qin, Dan Kou, and Yuanhe Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02271 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Environmental Science & Technology

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Magnitude and pathways of increased nitrous oxide emissions from uplands

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following permafrost thaw

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Guibiao Yang1,2, Yunfeng Peng1, Maija E. Marushchak3, Yongliang Chen1, Guanqin

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Wang1,2, Fei Li1,2, Dianye Zhang1,2, Jun Wang1,2, Jianchun Yu1,2, Li Liu1,2, Shuqi

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Qin1,2, Dan Kou1,2 and Yuanhe Yang1,2*

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1.

State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

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2.

University of Chinese Academy of Sciences, Beijing 100049, China

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3.

Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio 70211, Finland

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*

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10-62836632, E-mail: [email protected]

Corresponding author: Dr. Yuanhe Yang, tel.: + 86 10-62836638, fax: + 86

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1

ACS Paragon Plus Environment


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ABSTRACT: Permafrost thawing may release nitrous oxide (N2O) due to large N

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storage in cold environments. However, N2O emissions from permafrost regions have

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received little attention so far, particularly with respect to the underlying microbial

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mechanisms. We examined the magnitude of N2O fluxes following upland

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thermokarst formation along a 20-year thaw sequence within a thermo-erosion gully

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in a Tibetan swamp meadow. We also determined the importance of environmental

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factors and the related-microbial functional gene abundance. Our results showed that

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permafrost thawing led to a mass release of N2O in recently collapsed sites (3 years

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ago), particularly in exposed soil patches, which presented post-thaw emission rates

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equivalent to those from agricultural and tropical soils. In addition to abiotic factors,

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soil microorganisms exerted significant effects on the variability in the N2O emissions

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along the thaw sequence and between vegetated and exposed patches. Overall, our

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results demonstrate that upland thermokarst formation can lead to enhanced N2O

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emissions, and that the global warming potential (GWP) of N2O at the thermokarst

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sites can reach 60% of the GWP of CH4 (vs. ~6% in control sites), highlighting the

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potentially strong non-carbon (C) feedback to climate warming in permafrost regions.

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TOC Art

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Key-words: climate warming; denitrifiers; microbial functional genes; N2O emission;

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non-carbon (C) feedback; thermokarst; Tibetan Plateau

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Introduction

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Permafrost regions account for a large proportion of the global carbon (C) reservoirs,

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due to the slow microbial decomposition of soil organic matter under cold

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conditions1-3. The large amount of organic C stored in permafrost soils may represent

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increasingly relevant sources of methane (CH4) and carbon dioxide (CO2) to the

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atmosphere under global warming, which is more pronounced at high-latitudes than

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the global average4-7. The effects of permafrost thaw on gaseous C release have been

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extensively studied to evaluate the magnitude of permafrost C climate feedback4, 7-12.

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However, the large amount of nitrogen (N) stored in permafrost soils, which is a

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potential source for another greenhouse gas, nitrous oxide (N2O)8,

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ignored. Understanding N2O emissions from thawing permafrost is particularly

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critical because it has a high global warming potential (GWP), which is ~300-fold

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greater per unit mass than that of CO2 over a 100-year horizon22, and N2O is also the

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most important substance leading to ozone depletion in the stratosphere23.

13-21

, is often

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Permafrost thaw, leading to either gradual active layer deepening or often abrupt

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development of thermokarst landforms24, may increase N2O emissions due to the

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unlocking of vast N stocks stored in frozen soils. However, current experimental

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evidence are mainly derived from lowland permafrost ecosystems14,

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observations are available for upland ecosystems. Upland permafrost landscapes

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prone to thermokarst formation cover as much as 0.91×106 km2, or ~5% of the

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permafrost regions24 and belong to an important category of permafrost thawing 3


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, and limited


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around the world. More importantly, compared with lowlands which tend to be

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waterlogged following thermokarst formation25, thermokarst subsidence in upland

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permafrost usually results in intermediate or even lowered soil moisture26, 27 and the

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formation of adjacent aerobic and anaerobic soil environments8. These intermediate

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soil moisture conditions may promote coupled nitrification (oxidation of ammonia to

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NO3-) and denitrification (anaerobic respiration using NO3- as an electron acceptor),

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which may lead to high N2O production and emissions15, 28. Moreover, disturbances of

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the vegetation cover may also promote N2O emissions via improved nutrient

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availability for soil microbes in the absence of plant uptake15, 17. In support of these

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deductions, elevated N2O concentrations in the soil have been observed in upland

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thermokarst features8. However, direct field evidence of post-thaw N2O emissions in

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upland thermokarst remains unavailable.

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The escape of N2O from soils to the atmosphere after permafrost thaw is the result of

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the balance of multistep processes related to nitrification and denitrification29, and

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these processes are primarily driven by microbial pathways30. Multiple specialized

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microbial groups are responsible for nitrification-mediated (ammonia oxidizing

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archaea

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microorganisms). Further, the denitrification pathway is dependent on nitrification for

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the supply of nitrate, the initial electron acceptor for denitrification. Soil abiotic

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properties indirectly influence N2O flux via regulating microbial abundance and their

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activities31. Although the importance of plant and abiotic factors in the regulation of

and

bacteria)

and

denitrification-mediated

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pathways

(denitrifying

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N2O fluxes has been widely examined in permafrost areas (e.g., absence of plant N

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uptake, favorable moisture content and pH, low soil C:N ratios and relatively high

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inorganic N concentrations)13-15,

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directly associated with N2O production under permafrost thaw remain lacking19.

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, investigations of the microbial communities

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The Tibetan permafrost region, covering 67%, or ~1,500,000 km2, of the plateau

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area32, has experienced significant climate warming and consequential permafrost

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thawing over the past decades. The alpine swamp meadows, which contains a high

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soil N density similar to that in the northern circumpolar permafrost region (Fig. S1),

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offer an opportunity to examine the changes in N2O emissions following permafrost

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collapse together with the underlying mechanisms. Here, we measured the in situ N2O

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flux along a thermo-erosion gully in an alpine swamp meadow underlain by

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permafrost. The abiotic factors and microbial functional gene abundance were

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determined to gain a mechanistic understanding of how thermokarst formation affects

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N2O emissions. We hypothesize that 1) upland thermokarst formation enhances N2O

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emissions because of the mobilization of permafrost N and reduced plant N uptake,

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co-occurring with favorable moisture content, and that 2) thaw-induced changes in

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N2O flux are controlled by interactions between abiotic factors and soil

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microorganisms.

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Materials and Methods

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Experimental site 5



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The study site is an alpine swamp meadow underlain by discontinuous permafrost,

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located on the northeastern Tibetan Plateau, China (N 37°28′, E 100°17′, ~3900 m

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a.s.l; Fig. S2)33. The region is characterized by a wet alpine climate with a mean

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annual air temperature of -3.3°C and a mean annual precipitation of 460 mm. The

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active layer, which is affected by seasonal thawing, is ca. 90 cm thick as measured by

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a GSSI georadar (SIR-20, Laurel, Santa Clara, USA) and validated by manual

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measurements by a thaw probe. Vegetation at the site is mainly dominated by

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graminoids, such as Kobresia tibetica, K. royleana and Carex atrofuscoides. The soil

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in this area has a silty loam texture with 2.0% clay, 53.7% silt, and 44.3% sand. The

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chemical properties of the 0–15 cm soil layer are as follows: soil organic C, 19.4%;

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total N, 1.5%; bulk density, 0.27 g/cm3; and pH, 5.6.

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We measured the N2O flux along a thermo-erosion gully during two summers in 2015

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and 2016. The gully occurred in the early 1990s according to local residents and was

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~240 m long and approximately 2 m at the maximum depth at the time of

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measurements. In May 2015, four sites with an area of 15×20 m2 were established

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outside and inside the gully. The control site was an undisturbed area outside the

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thermo-erosion gully. The other three sites were dispersed at intervals of ~80 m from

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the head to the end along the gully, representing different thawing stages (early stage:

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collapse occurred 3 years ago; mid-stage: collapse occurred 12 years ago; and late

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stage: collapse occurred 20 years ago).

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N2O flux measurements

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Fluxes of N2O were determined using static chamber and gas chromatography

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techniques34. In 2015, ten vegetated patches were randomly chosen at the control site

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and at each of the three sites within the gully. A PVC ring (26 cm in diameter, 12 cm

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in height) with a collar was inserted into the soil to a depth of 10 cm. Because

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thermokarst formation often creates a mosaic of vegetated and exposed patches8 with

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potentially different N2O emission rate13,

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exposed patches inside the gully in 2016. The N2O flux was measured from 5

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positions in the exposed patches in each site due to their smaller proportion of the

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feature area. During gas collection, an opaque cylindrical chamber (25 cm tall) was

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placed on the collar. The chamber was covered with insulation materials to avoid a

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rapid increase in chamber temperature and equipped with an electric fan for proper

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mixing of the headspace air. Gas samples (50 ml) were collected from the headspace

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at 10-min intervals (0, 10, 20, 30, and 40 min) and immediately injected into

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pre-evacuated air bags (Delin Inc., Dalian, Liaoning, China). Gas samples were then

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transported to the laboratory and analyzed for N2O concentration using a gas

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chromatograph (Agilent 7890A, Agilent Technologies Inc., Santa Clara, California,

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USA; for analytical methodologies, see Wang & Wang, 2003). The rate of N2O flux

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was calculated from the slope of the N2O concentration–time function. Gas samples

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were collected between 9:00 am and 12:00 pm; in 2015, ca. every 10 days in July and

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August, and in 2016 once in early July, thrice during consecutive days at the end of

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July, once at the end of August. In total, 2275 gas samples were analyzed to obtain the

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, we also measured the N2O flux in

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N2O fluxes. At the time of gas collection, the soil temperature and volumetric water

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content (VWC) at a 0–10 cm soil depth were measured in each patch adjacent to the

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PVC ring using a digital thermometer and a portable TDR-100 soil moisture probe

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(Spectrum Technologies Inc., Plainfield, IL, USA).

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Soil sampling and biochemical analyses

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To discern the mechanism underlying the thermokarst effects on N2O emissions, we

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collected soil samples from the four sites and measured the soil physico-chemical

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characteristics and functional gene abundance of denitrifying microbes. Six soil cores

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(2.5 cm in diameter and 15 cm in depth) were collected near each PVC ring and

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homogenized to gain one composite sample during the intensive flux measurement

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campaign at the end of July 2016. In total, 240 soil cores were collected (40 soil

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samples). The soil samples were passed through a 2-mm-mesh sieve after removal of

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roots and divided into three parts. The first part was stored at 4°C to determine the soil

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nitrate (NO3 ) concentration. The second part was air-dried to measure the soil pH, and

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the third part was stored at -80°C to quantify the key microbial functional gene

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abundances. The soil NO3- concentration was determined using a flow injection

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analyzer (Autoanalyzer 3 SEAL; Bran and Luebbe, Norderstedt, Germany) after

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extracting fresh soil with 2 M KCl at a soil to solution ratio of 1:5 (v/v). Soil pH was

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measured in a soil water mixture (dry soil to deionized water ratio of 1:5 (v/v)) with a

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pH electrode (PB-10, Sartorius, Germany).

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For the soil microbial functional gene quantification, the present study only focused on

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denitrifying microorganisms because the soil water pore space (WFPS) was between

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62.6% and 84.1% across treatments and growing seasons. Under this condition,

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microbial denitrification is considered the predominant pathway of N2O production35.

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Here, we quantified the abundance of genes that encoded nitrite reductase (nirK and

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nirS) and nitrous oxide reductase (nosZ). For the analysis, DNA was extracted from

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wet soil (0.4 g) using the Power Soil® DNA Isolation Kit (MoBio Laboratories,

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Carlsbad, CA, USA) according to the manufacturers’ instructions. The DNA quality

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was checked with a NanoDrop-2000 (Thermo Fisher Scientific, Madison, WI, USA),

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which was assessed based on the absorbance values at 260/280 nm and 260/230 nm. A

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quantitative real-time polymerase chain reaction (qPCR) analysis was performed in

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triplicate on a StepOne Plus Real-Time PCR system (Applied Biosystems, Inc., CA,

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USA) in 20 µl of reaction mixture containing 10 µl of SYBR premix Ex Taq (Tli

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RNaseH Plus), 0.4 µl forward and reverse primers, 0.4 µl of ROX Reference Dye

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(50×), 6.8 µl of DNA-free water and 2 µl of fivefold diluted template DNA. The

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amplification conditions, reading temperature, primer pairs, number of cycles and

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references used to quantify the abundance of the nirK, nirS and nosZ genes are

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summarized in Table S1. For quantification, standard curves were constructed with a

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known concentration of purified plasmid DNA carrying the cloned portion of the

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corresponding gene with r2 > 0.95. The abundances of the nirK, nirS and nosZ genes

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are presented as a copy number per gram of dry weight (copies g-1 dw).

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Statistical analysis

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All data were checked for normality before the analysis. The N2O flux data were

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log-transformed to meet the assumption of a normal distribution. To assess the effects

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of thermokarst formation on N2O fluxes, a repeated-measures ANOVA was used to

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compare the differences in the N2O flux among the thawing stages and between

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vegetated and exposed patches, with the thawing stage or patch type as the

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between-subject factor, and the sampling date as the within-subject factor. One-way

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ANOVA were employed to evaluate the differences in seasonal means of the N2O flux,

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soil temperature and VWC as well as the soil pH, NO3 concentration, and the

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microbial functional gene abundance associated with denitrification (nirK, nirS, nosZ)

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among the thawing stages and between vegetated and exposed patches. We used post

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hoc tests (Tukey’s HSD) to test whether the above mentioned parameters exhibited

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significance at the level of P

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