Effects of mire disturbance by Sika deer on nitrogen fixation and denitrification in Ozegahara Mire, Japan

ABSTRACT

Ozegahara Mire is the largest alpine mire in Japan, and it has been preserved as the precious peatland ecosystem under oligotrophic condition. However, after the late 20 century, the mire has been exposed to ecosystem disturbance by invaded Sika deer. Browsing and digging of the mire vegetation by Sika deer have given considerable impacts on not only vegetation but also biochemical property of the peat soil, resulting in the changes in microbiological activities and nutrient dynamics. However, little information has been provided about the effect of deer disturbance on nitrogen (N) dynamics in the mire. This study aims to provide information about the effects of deer disturbance on nitrogen dynamic including N₂ fixation and denitrification in Ozegahara Mire. Soil samples were collected at 6 plots on 2 sites with and without mire disturbance by Sika deer at eastern Naka-Tashiro of Ozegahara mire in July 2018. Mineral N contents and other physicochemical properties of the soil samples were measured. N₂ fixation and denitrification activities of the soil samples were also measured at different incubation temperatures (15, 20 or 25°C) under either aerobic or anaerobic conditions by acetylene reduction activity (ARA) and inhibition methods, respectively. As results, the ARA in the disturbed sites were lower than those in the undisturbed sites and also lower under aerobic condition than anaerobic condition. Negative correlation was found between ARA and ammonium N in the soil (R² = 0.353, p< 0.05), suggesting that mechanical disturbance of the peat soils by deer enhanced mineralization of organic N to suppress N₂ fixation. ARA was increased by elevated incubation temperature in the undisturbed sites, while no change in the disturbed sites. Denitrification activities was not affected by the disturbance by Sika deer, while higher under aerobic condition than in anaerobic condition, and in higher temperature than lower temperature. Multi-regression analysis of ARA affecting factors revealed that ARA was significantly influenced by temperature, ammonium N and disturbance. These results indicate the marked influences of the mire disturbance by Sika deer on N₂ fixation and denitrification activities of the peat soils and N dynamics in Ozegahara Mire.

KEY WORDS:

• Denitrification
• N₂ fixation
• soil temperature
• peat soils
• Sika deer

1. Introduction

Ozegahara Mire is the largest alpine mire in Honshu (main island of Japan). This mire is located about 1400 m above sea level in the boundary of Gunma, Fukushima, and Niigata Prefectures, surrounded by mountains. The mire has been preserved as the precious peatland ecosystem under oligotrophic condition with high ecological diversity. This area was designed as a national park in 1953. It was also selected as a precious wetland by the Ramsar Convention in 2005 (Igarashi and Takatsuki 2008). In Ozegahara Mire, Scientific Research Campaigns were conducted almost every 20 years, and it was found in 4^th Scientific Research Campaign that flooding increased input of available phosphorus to some area in Ozegahara due to heavy rain more frequently happen recently, resulting increase densities of N₂-fixing plants (Myrica gale var. tomentotosa) (Shigeta et al. 2021). Under oligotrophic conditions like in Ozegahara, N₂ fixation is important nitrogen input to support vegetation and ecosystem (Shigeta et al. 2021). It is considered that, in flood prone area along the streams in Ozegahara, sediments carried by flooding water may supply significant amount of nutrients, while other area with less flooding, N₂-fixation by symbiotic N₂-fixing plants such as M. gale with N₂-fixating actinomyces and free-living microorganisms have significant role in N input from the atmosphere.

It has been discovered that Sika deer (Cervus nippon) started to invade Ozegahara in the 1990s (Naito and Kimura 1998). Bimonthly individual numbers of Sika deer crossing light sensing area in Ozegahara increased from about 50 in 2004 to more than 180 in 2019 (Figure S1; Ministry of Environment 2020). One of the reasons for increasing Sika deer is attributed to global warming (IPCC 2013) that makes their survivals possible over winter (Ministry of Environment 2020). Sika deer is potentially dangerous to such habitats because as their density and activities are increasing, and they can greatly affect plant communities. Sika deer in Ozegahara browse and dig in wetland to eat Menyanthes trifoliata rhizomes, and males of Sika deer which are in estrus dig up and rub against ground (Takatsuki 1989; Igarashi and Takatsuki 2008). It is known that browsing pressure by Sika deer have given considerable impacts on physicochemical properties of peat soil and vegetation (Pellerin, Huot, and Côté 2006: Takatsuki 2003). However, little information has been provided about the effect of deer disturbance on soil physicochemical and biological properties and nitrogen dynamics including N₂-fixing activity and denitrification activity in the mire. N is essential element for plant, so N dynamics, especially N₂-fixation and denitrification, and temperature sensitivities are important to preserve precious peatland ecosystem under oligotrophic condition, especially assessing impact of deer disturbance. This study aims at providing information about the effects of Sika deer disturbance on physicochemical and biological properties of the peat soils and the N dynamics in the Ozegahara Mire.

2. Materials and methods

We investigated physicochemical and biological properties of peat soil sample made mainly of Sphagnum materials taken from Ozegahara in Nikko National Park. Mean annual air temperature is 4.6°C and during the time of investigation season (June–September) was 12–18°C (Sakaguchi and Sohma 1982). We collected at 6 plots in 2 sites (A and B, with about 20 m distance) with (D1, D2, D3) or without (UD1, UD2, UD3) mire disturbance by Sika deer (3 replicates each) at eastern Naka-Tashiro area (36°55–57ʹN 139°12–15ʹE) of the Ozegahara mire. Disturbed sites are so-called nutaba in Japanese (Figure S2), but it is shown as ‘D’ in brown circles in Figures S3 and S4.

Soil samples (0–10 cm) were taken as 3 cores from each site with a trowel, brought back to the university laboratory in a cooler box to measure their physicochemical and biological properties. Moisture content, soil pH and EC (electric conductivity) were measured by gravimetric and electrode methods (TOA, CM-31P), respectively. The concentrations of ammonium and nitrate-N were measured in 1 M KCl extract by using the nitroprusside method (Anderson and Ingram 1989) and hydrazine reduction-naphthyl ethylene diamine method (Hayashi, Sakamoto, and Yoshida 1997), respectively.

The stable isotopic compositions of carbon (δ¹³C) and nitrogen (δ¹⁵N) of soil organic matter were determined using an isotope-ratio mass spectrometer (IRMS) connected to an elemental analyzer (Integra-CN, PDZ Europa, UK; Inubushi et al. 2020).

Soil acetylene reduction activity (ARA) was measured by ARA as C₂H₄ quantified with gas chromatograph (Shimadzu, GC-14B) equipped with a flame ionization detector (FID), and denitrification activity by acetylene block methods as N₂O quantified with another GC with an electron capture detector (ECD), respectively, both incubated at different incubation temperatures (15, 20 or 25°C) under either aerobic or anaerobic conditions (Japanese Society of Soil Microbiology 1992).

Statistical analysis was conducted to see the influence of environmental factors on soil anaerobic ARA by linear-regression analysis correlated with soil mineral N using Excel (Microsoft Office Professional Plus 2013) and multi-regression using CRAN R ver. 3.5.1 (R Core Team, Okada 2010), according to the formulae below.

(1) $Y=\alpha 0+\alpha 1F1+\alpha 2F2+\alpha 3F3+\alpha 4F4$(1)

(2) $Y=\alpha 0+\alpha 1F1+\alpha 2F2+\alpha 3F3$(2)

where Y indicates soil anaerobic ARA for dependent variable, while F1 as incubate temperature, F2 as soil ammonium N, F3 as soil nitrate N, and F4 as disturbance for explained variables, and α0 to α4 as factors, respectively.

3. Results

Soil moistures were basically high and pH low, similar as typical wetland (Inubushi et al. 2005), and both tended to be higher in the disturbed sites than in the undisturbed sites. Ammonium N was also higher in the disturbed sites, while no significant difference was found for soil EC and nitrate N contents (Figure 1).

Figure 1. (a) Soil moisture (%), (b) pH (left) and EC (right; mS m⁻¹) and (c) mineral N (upper: nitrate, lower: ammonium, mg N kg⁻¹ wet soil) contents in disturbed (D) and undisturbed (UD) sites.

The ARA in the disturbed sites were lower than those in the undisturbed sites and also lower under aerobic condition than anaerobic condition (Figure 2). Negative correlation was found between ARA and ammonium N in the soil (R² = 0.353, p< 0.05, Figure 3). ARA was increased by elevated incubation temperature in the undisturbed sites, while no significant difference in the disturbed sites in three temperatures.

Figure 2. ARA of disturbed (D) and undisturbed (UD) soils under aerobic (upper) or anaerobic (lower) conditions with different temperatures.

Figure 3. Correlation between ARA and ammonium N of disturbed (D) and undisturbed (UD) soils with different temperatures.

Denitrification activities was higher under aerobic condition than in anaerobic condition, while not affected by the disturbance by Sika deer, and mostly higher in higher temperature than in lower temperature (Figure 4). Stable isotope ratios of soil organic C and N were not significantly different between disturbed and undisturbed sites (Figure 5).

Figure 4. Denitrification activities of disturbed (D) and undisturbed (UD) soils under aerobic (upper) or anaerobic (lower) conditions with different temperatures.

Figure 5. Stable isotope ratios of disturbed (D) and undisturbed (UD) soils.

Temperature sensitivity, Q₁₀ (increase ratios of the activities from 15 to 25°C), for ARA under anaerobic conditions was 0.88 ± 0.72 and 3.21 ± 1.32 (mean ± standard deviation) in disturbed and undisturbed sites, respectively, while 1.75–2.76 for denitrification activities without significant influence of disturbance (Table 1).

Table 1. Q₁₀ (25/15°C) of ARA and denitrification activities of disturbed (D) and undisturbed (UD) soils under aerobic or anaerobic conditions

		ARA	Denitrification
		Anaerobic	Aerobic	Anaerobic	Aerobic
Site A	D1	1.68	-*	3.83	1.83
	D2	0.69	-	2.37	2.84
	UD1	3.93	10.05	3.01	1.72
Site B	D3	0.27	2.55	2.08	1.83
	UD2	4.01	-	2.54	1.82
	UD3	1.69	-	2.03	1.70

*ARA was below detection limit.

Multi-regression analysis of ARA indicated that main affecting factors were the disturbance, ammonium N and temperatures, in this order for the pooled values in the disturbed and undisturbed sites using formula (1), and also significantly affected by ammonium N in the undisturbed site, while no significance was found in the disturbed site using formula (2) (Table 2).

Table 2. Multi-regression analysis of affecting factors for anaerobic ARA in disturbed (D) and undisturbed (UD) soils

	Standardized partial regression coefficient				Determination coefficient
	Temperature	NH4^+-N	NO3^–N	Disturbance	R^2	p value
	(F1)	(F2)	(F3)	(F4)
Site (Formula^#)	α1	α2	α3	α4
D + UD (1)	0.248*	-0.298*	0.111^NS	0.502***	0.489	<0.01
D (2)	-0.227^NS	-0.176^NS	0.084^NS	-	-0.027	0.52
UD (2)	0.557^NS	-0.568**	-0.31^’	-	0.571	<0.01

^#Formula, see text, ***indicates p < 0.001, **p < 0.01, *p < 0.05, and ‘p < 0.1, respectively.

4. Discussion

In the undisturbed natural area, such N₂ fixation can be maintained, while in the disturbed area by Sika deer, N₂ fixation may be suppressed by increase of ammonium N derived from decomposition of physically damaged peat materials.

After Sika deer disturbance, it has reported that peat soil temperature increased due to direct sun light (Furusawa et al. 2006), so temperature sensitive denitrification may also be enhanced, while N₂-fixation in disturbed area is keep suppressed (Table 1), inducing changes in N dynamics and cause ecosystem disturbances. Lee, Suzuki, and Inubushi (2018) also reported that carbon decomposition is depend on in-situ temperature, resulting differences in CO₂ and CH₄ emission rates from brackish marsh in northern Honshu Island, Japan. These reports support that Sika beer disturbance also enhance organic matter decomposition by rising soil temperature and then affected also N dynamics in Ozegahara.

Yoh and Tsuruta (1998) reported that anaerobic ARA was 1 magnitude higher than aerobic ARA which was similar as our results, and had peaked at about 17 cm deep peat profile in Shimo-Tashiro, Ozegahara. Multi-regression analysis of anaerobic ARA showed that ammonium N and the Sika deer disturbance had strongest impacts to suppress the N₂-fixation activity.

Relatively higher δ¹³C and lower δ¹⁵N values were found for disturbed samples than undisturbed samples (Figure 5). Akagi and Osawa (2004) reported that sundew samples along wooden walkway tended to be higher δ¹³C and lower δ¹⁵N values than peat moss samples in Ozegahara, which indicated higher human (visitor) s’ impact on natural vegetation even in strictly protected environment like national park. Although no significance in δ¹⁵N values, lower values in disturbed sites suggested that effect of Sika deer disturbance might be caused by suppression of N₂ fixation, however still limited data in short term and surface soil layers to consider N dynamics. Longer and more detail investigation is necessary.

In conclusion, these results suggested that mechanical disturbance of the peat soils by Sika deer enhanced mineralization of organic N to suppress N₂ fixation. These results indicate the marked influences of the mire disturbance by Sika deer on N₂ fixation and denitrification activities of the peat soils and N dynamics in Ozegahara Mire.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

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Additional information

Funding

This work was a part of 4^th Scientific Research Campaign in Ozegahara, supported by the Ministry of The Environment, Japan.