Application of δ15N to trace the impact of penguin guano on terrestrial and aquatic nitrogen cycles in Victoria Land, Ross Sea region, Antarctica

Journal Pre-proofs Application of δ 15N to trace the impact of penguin guano on terrestrial and aquatic nitrogen cycles in Victoria Land, Ross Sea region, Antarctica Xueying Wang, Xiaodong Liu, Yunting Fang, Jing Jin, Libin Wu, Pingqing Fu, Huihui Huang, Huijun Zhang, Steven D. Emslie PII: DOI: Reference:

S0048-9697(19)34487-0 https://doi.org/10.1016/j.scitotenv.2019.134496 STOTEN 134496

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Science of the Total Environment

Received Date: Revised Date: Accepted Date:

28 June 2019 9 September 2019 15 September 2019

Please cite this article as: X. Wang, X. Liu, Y. Fang, J. Jin, L. Wu, P. Fu, H. Huang, H. Zhang, S.D. Emslie, Application of δ 15N to trace the impact of penguin guano on terrestrial and aquatic nitrogen cycles in Victoria Land, Ross Sea region, Antarctica, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv. 2019.134496

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Application of δ15N to trace the impact of penguin guano on terrestrial and aquatic nitrogen cycles in Victoria Land, Ross Sea region, Antarctica

Xueying Wanga, Xiaodong Liua,*, Yunting Fangb, Jing Jina, Libin Wuc, Pingqing Fuc, Huihui Huanga, Huijun Zhanga, Steven D. Emslied

aAnhui

Key Laboratory of Polar Environment and Global Change, School of Earth and Space Sciences,

University of Science and Technology of China, Hefei, Anhui 230026, China bCAS

Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese

Academy of Sciences, Shenyang 110016, PR China cInstitute

of Surface-Earth System Science, Tianjin University, Tianjin 300072, China

dDepartment

of Biology and Marine Biology, University of North Carolina Wilmington, 601 S. College

Road, Wilmington, NC 28403, USA *Corresponding Author, E-mail: [email protected]

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Abstract

Penguin colonies in Antarctica offer an ideal “natural laboratory” to investigate ecosystem function and the nitrogen (N) cycle. This study assessed the spatial distribution of penguin-derived N from guano and quantitatively assessed its impact on plant N utilization strategies in Victoria Land, Ross Sea region, Antarctica. Soil, moss, and aquatic microbial mats were collected inside and outside an active Adélie penguin (Pygoscelis adeliae) colony and analyzed for δ15N of total and inorganic nitrogen (NH4+-N and NO3–-N). The soil total nitrogen (TN), NH4+-N, and NO3–-N concentrations, as well as their δ15N values were significantly higher in guano-impacted areas than those in guano-free areas, verifying that guano is an important N source at and near penguin colonies. However, even far from the penguin colonies, soil δ15N values resembled those in penguin colonies, suggesting strong spatial impacts of penguin-derived N. The moss impacted by guano was more enriched in δ15N than in guano-free areas. The δ15N values of NH4+-N and NO3–-N in soils covered with moss revealed that the moss might prefer inorganic N in the absence of guano, while the dissolved organic N would become an important source for moss growing on ornithogenic soils. Aquatic microbial mat samples near penguin colonies were 15N-enriched, but 15N-depleted at upland sites. Keywords: Guano; Moss; Soil; Microbial mat; δ15N; East Antarctica

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1. Introduction

Nitrogen (N) is a key nutrient for ecosystem function and both regional and global N cycles have been of primary interest in environmental and ecological research (Gruber and Galloway, 2008; Hundey et al., 2016). The N cycle includes numerous N compounds with the most abundant form, atmospheric dinitrogen (N2), unavailable to most organisms. Reactive N (Nr), including ammonium (NH4+), ammonia (NH3), nitrate (NO3−), and N oxide (NOx) are biologically available and thus more important for the biosphere (Galloway et al., 2008; Fowler et al., 2013). With the increase of human activities including intensive use of fertilizer in agriculture, urbanization and fossil fuel combustion, the level of Nr has significantly increased globally (Galloway et al., 2008; Boyle 2017). As a result, the original pattern of the natural N cycle has changed considerably, which has caused many environmental issues, including aquatic eutrophication and soil acidification (Logan, 1983; Anderson et al., 2002; Lu et al., 2014). Most studies on the N cycle have focused on areas close to human activities, such as grasslands, forest, farmland, wetlands, and river and lake ecosystems (Baron et al., 2013; Sebilo et al., 2013). To our knowledge, there is a considerable amount of information on N cycling in remote areas such as the Arctic (Choudhary et al., 2016; González-Bergonzoni et al., 2017; Barthelemy et al., 2018). However, knowledge on N cycling in the Antarctic is still relatively lacking. Although far from human activities, Antarctic ecosystems are especially sensitive to global

3

climate change and human activities (Barrett et al., 2006). To increase knowledge on the process and mechanisms of the N cycle, and to better predict changes of this cycle in the future, large-scale investigations in Antarctica are warranted. The global distribution of seabird colonies is confined mainly to polar zones, especially in Antarctica and sub-Antarctic islands (Otero et al., 2018). There are millions of penguins that nest in ice-free coastal regions of Antarctica, where the biological transmission of nutrients from marine to terrestrial ecosystems via guano has a significant role in the nitrogen cycle in soils (Bokhorst et al., 2019). Previous studies have shown that penguin guano has a strong influence on soil properties and terrestrial bio-community structure (Tatur et al., 1997; Sun et al., 2000; Wang et al., 2007; Smykla et al., 2015). Guano contains ~15–20% N and 10% phosphorus (P) (Lorrain et al., 2017). These potentially limiting nutrients can greatly change soil properties and stimulate plant growth (Heindel et al., 2018). With accumulated guano, penguin colonies represent a typical area with concentrated sources of N and/or ammonia in the Antarctic and can act as biological ‘hotspots’ for lichens, moss, and algal diversity (Riddick et al., 2012, 2017; Huang et al., 2014; Smykla et al., 2018). Thus, penguin colonies in Antarctica are ideal “natural laboratories” to investigate N cycles and their influence on terrestrial community structure. In previous studies, the emission of atmospheric NH3 volatilized by uric acid from guano has been the focus of the N cycle in penguin areas (Riddick et al., 2016, 2018). Many studies also have investigated the impact of penguin guano on lichen and 4

plant communities (Beyer et al., 2000; Leishman and Wild, 2001; Smykla et al., 2007). However, the spatial extent of NH3 derived from colonies and its impacts on plant N uptake is still poorly understood (Crittenden et al., 2015). The dispersion model of NH3 indicates that penguin colonies have the potential to produce a large impact zone (beyond 10 km) in surrounding areas (Crittenden et al., 2015). The latest study in the Antarctic Peninsula demonstrated a spatial impact of marine-derived N of >1,000 m from the immediate colony borders (Bokhorst et al., 2019). To obtain a deeper understanding of the N cycle and its mechanisms in penguin areas, investigation on the spatial extension of penguin-derived N and its impact on plant N utilization strategies is still required. The natural abundance of 15N has been widely used as a powerful in situ tracer of biological processes through their capacity to alter isotopic ratios (Wada et al., 1981; Liu et al., 2006; Li et al., 2010; Pfister et al., 2014). Nevertheless, due to the complexity of the nitrogen cycle, the interpretation of natural abundance data can be problematic and requires careful consideration. Due to an often high trophic position in seabirds and preferential volatilization of 14N from guano, guano is enriched in 15N (Mizutani et al., 1985). Thus, δ15N can be a useful tool to trace seabird-derived N. Many studies have shown that seabirds provide N sources in their surrounding environment, leading to high δ15N values in plants growing near their colonies (Mizota and Naikatin, 2007; Durrett et al., 2014; Lorrain et al., 2017). For example, δ15N was found to be higher in soil and plants at seabird-impacted sites compared 5

with seabird-free sites in the Antarctic (Crittenden et al., 2015), Arctic (Zmudczyńska-Skarbek et al., 2015; González-Bergonzoni et al., 2017), New Zealand (Harding et al., 2004; Hawke and Newman, 2007), Hawaii (Honig and Mahoney, 2016), Scotland (Callaham et al., 2012), Florida (Irick et al., 2015), Fiji (Mizota and Naikatini, 2007), New Caledonia (Lorrain et al., 2017), and Chagos Archipelago (Graham et al., 2018). In recent years, the isotopes of inorganic N, including δ15N of NH4+, δ15N and δ18O of NO3− have been widely adopted in identifying their source and movement in various environments (Elliott et al., 2009; Chang et al., 2016). In particular, δ15N and δ18O of NO3− can be used to track the source of nitrate due to the distinct isotopic characteristics of the main sources of nitrate, such as atmospheric deposition, chemical fertilizers, animal feces, and nitrate derived from nitrification (Li et al., 2010). Furthermore, δ15N and δ18O of NO3− can provide useful information to identify denitrification processes because

15N

and

18O

are preferentially enriched in

residual nitrate during this process (Mizota, 2009). However, most of studies in Antarctica have focused primarily on the isotope analysis of total N (TN) instead of the inorganic fractions (Nie et al., 2014). Uric acid (organic N) is the main constituent of guano and can rapidly mineralize into bioavailable forms, such as NH4+ and NO3− (inorganic N) (Lindeboom, 1984). Some previous studies have shown that stable isotope analysis of inorganic N can be used to trace important information on ecological aspects in remote regions of Antarctica (Wada et al., 1981, 1984, 2012). Thus, the combination of total and inorganic N isotope analysis may provide new 6

insight in N cycle process associated with Antarctic penguin colonies. Here, we determined δ15N of TN and inorganic N (NH4+ and NO3−) in soil, and δ15N of TN in moss and aquatic microbial mats, inside and outside Adélie penguin (Pygoscelis adeliae) colonies in Victoria Land, Ross Sea region, Antarctica. The goal of this study was to assess the spatial extent of penguin-derived N and its influence on plant N utilization strategies. Our results expand on existing knowledge on the impact of penguins on N cycles in remote Antarctica ecosystems, allowing predictions of their fate with global environmental change.

2. Materials and methods

2.1. Study sites description

Field investigation was conducted at several ice-free areas, with and without penguin colonies, in Victoria Land during austral summer 2015-2016 (December 2015 to February 2016; Fig. 1A). There are numerous raised beaches and islands with modern active or abandoned Adélie penguin colonies along the Victoria Land coast (Baroni and Orombelli, 1994a; Emslie et al., 2007). The climate is frigid and is a typical coastal climate for the Antarctic continent with temperatures and precipitation at low levels (Smykla et al., 2011). The average annual temperature ranges from −17 to −19 ℃ in the central part of Victoria Land and the precipitation is always from snow with averages of 100–200 mm per year, and the vegetation is comprised entirely 7

of cryptogams as vascular plants are absent (Smykla et al., 2011). Detailed ecological and geological surveys and sampling were conducted at active and abandoned penguin sites in Victoria Land. Sampling areas include Cape Irizar, Inexpressible Island, North Adélie Cove, Campo Icarus and Cape Hallett (Fig. 1A). Here, Inexpressible Island (74°54'S, 163°43'E) is the focus of this study and is located in Terra Nova Bay, Northern Victoria Land (Fig. 1B). The west end of the island is adjacent to the Nansen Ice Sheet, while the northeast side is connected to the Hell's Gate Ice Shelf. Inexpressible Island is sensitive to climate change and is strongly influenced by continental glaciers and ice sheets, making this site an ideal place for conducting research on the interaction between paleoecology and climate change (Baroni and Orombelli, 1994b; Bargagli et al., 2007). It has numerous raised beaches at different elevations (Baroni and Hall, 2004). A large number of abandoned Adélie penguin colonies also occur on or near these raised beaches and there is an active colony of more than 20,000 pairs occupying ice-free terrain surrounding Seaview Bay (Lynch and LaRue 2014; He et al., 2017). Microbial mats (or freshwater algal mats) are widely distributed in ponds near penguin colonies in the Ross Sea region due to the nutrient input from their guano. Meteorological data indicate that the interannual variability of the climate at Inexpressible Island is significant (Ding et al., 2015). The monthly average temperature is below 0°C, and the annual average ranges from −15.3 to −18.7℃. In addition, precipitation on the island is relatively low. The strong downwind and the constant wind direction are typical characteristics of this island. 8

2.2 Sample collection and preparation

Bare soil, soil associated with moss (hereinafter referred to “moss-growth soil”), moss, and aquatic microbial mat samples were collected in penguin and non-penguin areas. A total of 55 moss samples including 29 guano-free and 26 guano-impacted were collected at Cape Irizar, Inexpressible Island, North Adélie Cove, Campo Icarus and Cape Hallett (Fig. 1A). We analyzed a total of 31 moss-growth soil samples as some of the soil associated with moss were insufficient for analysis. Other samples (bare soil and aquatic microbial mats) were all collected at Inexpressible Island. All detailed sample information used in this study is given in the SI Appendix. Moss samples were sent to the Institute of Botany, Chinese Academy of Sciences for the species identification. A total of 13 bare soil samples were collected from abandoned ancient penguin colonies (marked as “A”, referring to an abandoned penguin colony where there is no penguin activities now), modern active penguin colonies (marked as “M”, referring to the current presence of a large number of penguins), and from fellfield areas far outside the penguin colonies (marked as “F”, referring to sites without any trace of penguin activities, with the farthest site estimated to be at least 1 kilometer from the colony) at Inexpressible Island (Fig. 1C). There are many small ponds or lakes present at Inexpressible Island. These ponds were formed by glacial erosion and are mainly supplied by melting snow and local precipitation. Pond water is primarily lost through 9

evaporation with no visible outlets, and most of the pond ice was absent during sampling (December to February) (Zhao et al., 2015). A large number of floating microbial assemblages were present on the surface of the ponds with dried microbial mats abundant at the shoreline. A total of 12 freshwater microbial mat samples were collected along a gradient beginning near the active penguin colony and extending to upland fellfield areas on the island (Fig. 1C). According to field observations, there was no obvious penguin activity at the microbial mat sampling sites. All the collected samples were stored in clean polyethylene plastic bags or sealed bottles and then transported to the laboratory and kept at −20°C until they were analyzed. Before analysis, soil samples were freeze-dried and passed through a 2 mm mesh sieve to remove coarse fragments. The sieved soils were divided into two parts. One part was reserved for subsequent measurement of NH4+ and NO3− concentrations. The other part was ground to a 200-mesh size using a mortar and pestle, and then stored at −20°C in clean polyethylene plastic bags. Moss and aquatic microbial mat samples were acid-treated (1 M HCl) to remove surface impurities and freeze-dried for subsequent homogenization.

2.3 Chemical and stable isotope analyses

P is a typical bio-element of penguin-derived nutrients, and soil P, TOC, and TN contents are usually much higher in penguin areas than the background values from control sites (Liu et al., 2013b). To test whether soil nutrient levels are significantly 10

affected by guano, soil concentrations of P, TOC, and TN were determined. A total of 0.1 g of each soil sample was digested with HNO3, HCl, and HClO4 in a microwave-assisted digestion, and finally, the P concentration was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES: 2100DV, Perkin Elmer, Waltham, MA, USA) (Wu et al., 2018). The soil TOC concentration was determined by potassium dichromate oxidation–volumetric method (Wu et al., 2017, 2018). The bulk carbon (C) and N concentrations of moss, soil, and microbial mat samples were measured by a FLASH 2000 HT Elemental Analyzer (Thermo Fisher Scientific) with an analytical precision of 0.01% (Wu et al., 2017, 2018). Briefly, about 10 mg of each dried sample was placed in a tin cup, wrapped to compact it, and then analyzed. The total nitrogen δ15N values of bare soil, moss-growth soil, moss and microbial mat samples were analyzed using an isotope ratio mass spectrometer (IRMS: MAT 253). The determination of δ15N values in soil requires powdered samples with no pre-treatment. Samples and standards were completely combusted and gases were separated by a “purge and trap” adsorption column and sent to the IRMS for analysis (Wu et al., 2018). The expression of stable isotope abundance is δ notation as the deviation from standards in parts per thousand (‰): δ15N = [(R sample / R standard) − 1] × 1000 (‰), where R represents the

15N/14N

ratio and the R standard value was based

on atmospheric N (N2-atm). Analytical precision for δ15N was better than ±0.2‰. To further study the effect of penguin-derived N on moss nitrogen utilization 11

strategies, we analyzed moss-growth soil NH4+-N and NO3–-N concentrations as well as soil δ15N-NO3−, and δ15N-NH4+ values. A 2 M KCl solution was used to extract soil NH4+ and NO3−. Briefly, about 10 g of sieved soil sample was extracted with 50 mL of 2 M KCl solution (Wang et al., 2014). The concentrations of NH4+and NO3− in KCl solution were then measured by Smart Chem 200 Discrete Chemistry Analyzer to obtain the concentrations of NH4+ and NO3− in soils (Esfandbod et al., 2017). In view of the large amount of sample required for inorganic N analysis, we analyzed a total of 19 moss-growth soil samples with sufficient amount for inorganic nitrogen. The isotope analysis of soil NH4+ and NO3− included δ15N of NH4+ and δ15N and δ18O of NO3−. Analysis of δ15N-NH4+ was by micro-diffusion method because it can avoid interference of organic N (Zhang et al., 2015). Briefly, NH4+ was extracted and then it was oxidized to NO2− by alkaline hypobromite (BrO−), and finally reduced to N2O by hydroxylamine (NH2OH) (Liu et al., 2014). Analysis of δ15N and δ18O of NO3− was completed by the denitrifier method (Sigman et al., 2001). This method is based on isotope analysis of nitrous oxide (N2O) produced by denitrifying bacteria which lacks N2O- reductase activity. Reagent blanks and international standards of NH4+ (IAEA N1, USGS 25 and USGS26) and NO3− (IAEA N3, USGS 32, USGS 34 and USGS 35) were used in every batch of analyses, and NaNO2− was used as the secondary standard. The isotopic signatures of N2O were determined by an IsoPrime 100 continuous flow isotope ratio mass spectrometer connected to a trace gas (TG) pre-concentrator (IsoPrime, CheadleHulme, UK). The expression of δ15N is described 12

above, and δ18O= [(Rsample/Rstandard) −1] × 1000 (‰), where R is the18O/16O ratio and R standard was based on standard mean ocean water (SMOW). The analytical precision for isotopic analyses (δ15N and δ18O of NO3−, and δ15N of NH4+) was better than 0.3‰. Additional details are provided in our previous study (Wu et al., 2018).

2.4 Data Analysis

To compare whether the soil N contents and δ15N values inside the penguin colony are significantly different from those outside the colony, we used SPSS (version 24.0) to compare mean soil N contents or δ15N values among the different sampling sites. Statistical significance was inferred for P<0.05. To further understand the relationships among N-related indicators, correlation analysis of soil P, TOC, N, NH4+ and NO3− concentrations, as well as δ15N-TN, δ15N-NH4+, δ15N-NO3−, and δ18O-NO3− values from F, M and A sites was conducted using SPSS (version 24.0).

3. Results

3.1. Soil N concentrations and δ15N values

The lowest concentrations of P, TOC and TN were found in fellfield soil samples far away from the penguin colony (marked as “F”), while the highest values were present in those from a modern active penguin colony (marked as “M”), followed by an abandoned ancient penguin colony (marked as “A”) (Fig. 2A). There were much 13

higher concentrations of NH4+-N than NO3–-N in M soils (Fig. 2C), indicating that these comprise the main form of soil inorganic N. The proportion of NH4+-N to TN was highest in F soils (CNH4+-N/CTN = 77.8 ± 19.9%, n=3), followed by M (CNH4+-N/CTN= 22.7 ± 12.3%, n=7) and A soils (CNH4+-N/CTN=0.63 ± 0.26%, n=3). Statistical analysis showed that the mean soil TN and NH4+-N concentrations were significantly higher in M than those in F soils (t-test, P<0.05) and A (P<0.05). The mean δ15N-TN of soil samples collected from the F, M and A sites ranged from 16.4 to 20.5 ‰, and soil δ15N-TN in M sites were highest (Fig. 2B). The δ15N-NH4+ values were lowest in A soils (12.2 ± 11.1 ‰) compared to F (δ15N-NH4+ = 37.5 ± 4.7 ‰, t-test, P<0.05) and M soils (δ15N-NH4+ = 40.2 ± 5.8 ‰, t-test, P<0.05). Soil δ15N-NO3– values showed large variation with the means in F, M and A soils at 3.9 ± 5.1 ‰, 10.0 ± 14.1 ‰, and 7.1 ± 10.7 ‰, respectively, and they were obviously lower than δ15N-NH4+ values at all sampling sites. Statistical analysis showed that soil δ15N-TN and δ15N-NH4+ values in M were significantly higher than those in A soils (Fig. 2B, t-test, P<0.05), but not significantly differ from those in F soils (Fig. 2B, t-test, P>0.05). In addition, the mean δ18O-NO3– value was significantly higher in M (13.7 ± 6.0 ‰) than in F (−0.4 ± 3.3 ‰, t-test, P<0.05) and A soils (−11.6 ± 7.1 ‰, t-test, P<0.05, Fig. 2D). Correlation analysis indicated that soil P concentrations at F, M and A sites were positively correlated with the concentrations of soil TOC, TN, NH4+-N and NO3−-N (Table 1). In addition, there were positive correlations between soil P concentrations 14

and δ18O-NO3− values, and between soil δ15N-NH4+ and soil δ18O-NO3− values (Table 1).

3.2. Moss and moss-growth soil δ15N

Most of the moss species were Syntrichum magellanica (Mont.) R.H. Zander, with a few identified as Bryum pallescens Schwagr and Bryum argenteum Hedw. P, TOC, and TN concentrations in the moss-growth soil as well as bulk C and N concentrations of moss inside and outside penguin colonies in ice-free regions of Victoria Land are in Fig. 3. It was obvious that the moss-growth soil affected by guano had higher concentrations of C, N and P. Similarly, moss grown in ornithogenic soil had higher concentrations of C and N (t-test, P<0.05). The mean δ15N value of moss growing in guano-impacted areas was significantly higher than that in guano-free areas (t-test, P<0.05). The results of isotopic analysis on inorganic nitrogen showed that the moss-growth soil impacted by guano had higher δ15N-NH4+ and δ15N-NO3– values (Fig. 4). For moss-growth soil not impacted by guano, the δ15N-TN values of moss were lower than those of soil. However, there were no significant differences between moss δ15N-TN and soil δ15N-NH4+ values, or between moss δ15N-TN and soil δ15N-NO3– values. For moss-growth soil impacted by guano, there was no significant difference between moss δ15N-TN and soil δ15N-TN, but the δ15N-TN values of moss were significantly higher than δ15N-NH4+ values of moss-growth-soil (t-test, P<0.05). There 15

was no significant difference between moss δ15N-TN and soil δ15N-NO3– values. Correlation analysis showed that δ15N-TN values of moss-growth soil were positively correlated with soil P concentrations (r=0.840, n=31, P<0.01), and moss δ15N-TN values were positively correlated with soil δ15N-TN values (r=0.853, n=31, P<0.01), but there was no significant correlation between moss δ15N-TN values and soil δ15N-NH4+, and δ15N-NO3– values.

3.3. δ15N in aquatic microbial mat

The TN concentration and δ15N-TN values of aquatic microbial mats in the ponds and lakes collected along a gradient from the active penguin colony to the upland fellfield area were illustrated in Fig. 5, and the results showed that the N concentration decreases with the altitude and distance away from the penguin colony, while the δ15N values became more negative. We found a strong regression correlation between the distance (sampling ponds to the center of the modern penguin colony) and microbial mat δ15N values, while the correlation between the relative elevation of sampling ponds to the modern penguin colony and microbial mat δ15N values was relatively weak (Fig. S1, SI Appendix). On the whole, the δ15N values of aquatic microbial mats can be categorized into three statistically significant types: 15N-enriched values (10.4 ± 1.7 ‰) were found at sites closer to the active penguin colony, and 15N-depleted values were −0.5 ‰ at the middle distance from the colony. In contrast to these two situations, microbial mats in 16

the fellfield sites on an upland plateau had 15N-depleted signatures of −11.7 ± 1.0 ‰ (Fig. 5).

4. Discussion

4.1. N sources on Inexpressible Island

In the absence of biological activity, the ability of non-biological processes to transport nutrients on Inexpressible Island is very limited. Therefore, environmental media such as soil on the island exhibit an oligotrophic status under initial conditions. However, penguin activities on the island transport a large amount of nutrients such as N and P, and this is very important for driving the material cycle in ecosystem function. It is likely that there are several possible N sources on Inexpressible Island: biological transportation (guano), atmospheric dry and wet deposition, and biological fixation. Nitrogen fixation is not thought to widely occur in Victoria Land soils, and biological transformations of N have been reported for soils and sediments in hydrological margins of Dry Valley streams and lakes (Barrett et al., 2002, 2006). Here, we use P content to represent the contribution of penguin guano to the soil because P is a typical bio-element of guano (Liu et al., 2013b). The positive correlations between bare soil P and N (TN, NH4+-N and NO3–-N), and between bare soil P and soil TOC (Table 1) suggest that guano is the most important source of nutrients on Inexpressible Island, and penguin-derived N controls the soil N content 17

and distribution in the area. There were no significant differences in δ15N-TN or δ15N-NH4+ values between M and F soils (Fig. 2B), indicating that the N source in F soil is mainly from penguin guano. These results showed that the volatile ammonia from penguin guano can cause significant impact on the N source of bare soils far away from the active penguin colony at Inexpressible Island. Indeed, previous research has shown that the penguin colonies have the potential to produce a large impact zone (beyond 10 km) in surrounding areas, and the spatial impact zone of penguin-derived N is considerable (Crittenden et al., 2015). Considering the small size of our analytical samples in the present study, we suggest that further research is needed to expand the sampling zone and quantitatively calculate the spatial extent of volatile ammonia from penguin guano at Inexpressible Island, even in Victoria Land. We found that bare soil NH4+-N concentrations were much higher than NO3–-N concentrations at all sampling sites (Fig. 2C). Low concentrations of NO3–-N indicates limited nitrification or rapid denitrification processes in soils (Mizota, 2009). The concentrations of different N forms in soil collected from F, M and A areas indicated that NH4+-N was the main N form in the soils far away from the penguin colony, and organic N was the main N form of soils in the abandoned penguin colonies. Both inorganic and organic N were equally important forms in the soil samples collected from the active penguin colony (details are provided in SI Appendix). Based on these results, we speculate that in soil distant from a penguin colony, NH4+-N will mainly source from deposition of volatile ammonia from penguin guano in surrounding areas. 18

Indeed, previous studies have shown that penguin colonies are the major source of NH3 emissions to the atmosphere (Anderson and Polis, 1999; Crittenden et al., 2015). As guano was the main N source in the soil of the active penguin colony, once deposited, strong NH3 volatilization takes place rapidly. Then, the mineralization and nitrification process would result in both inorganic and organic N as equally important forms in soils for N biogeochemistry at the active penguin colony. Therefore, an active penguin colony is the most important area for the N cycle. In the soil of an abandoned ancient penguin colony, the very low concentration of NO3–-N and NH4+-N suggest that a large amount of inorganic N is lost through NH3 volatilization, denitrification and leaching. Generally, less fractionation occurs during mineralization of organic N in guano diagenesis (0 to –5‰), while in nitrification, denitrification, and ammonia volatilization, a large isotopic fractionation occurs (Robinson, 2001). Soil δ15N-NO3– values were much lower than δ15N-NH4+ values in M and F soils (Fig. 2B), suggesting large kinetic isotopic fractionation during nitrification of NH4+-N into NO3–-N. The mean soil δ15N-NH4+ value was higher than δ15N-TN at the active penguin colony (Fig. 2A), indicating that a strong NH3 volatilization leaves behind 15N-enriched NH4+. The mean soil δ15N-NH4+ value was relatively low in abandoned ancient compared with active penguin colonies (Fig. 2B), likely due to persistent leaching of NH4+ enriched with heavy

15N

in ancient ornithogenic soil and then deposited in the

catchment or ponds. Indeed, the low concentration of NH4+-N in the soil of an 19

abandoned penguin colony indicates that a large amount of NH4+-N was lost through runoff and other process (Fig. 2C), and also our previous results demonstrated that the heavily guano-impacted sediments in the pond around the active penguin colonies had very high δ15N values and NH4+ contents (Nie et al., 2014). The mean δ18O-NO3– value was significantly higher in M than that in F and A soils (Fig. 2D), with positive correlations between soil P concentrations and δ18O-NO3− values (Table 1). There are two possible interpretations for such high δ18O-NO3– values in the soils at an active penguin colony. First, with strong denitrification present in M soil, guano input increased soil organic matter content, and prompt denitrification could occur under the supply of an electron donor such as organic matter (Mizota, 2009). Previous studies have shown that high N2O emissions occurs in active penguin colonies and denitrification was the main source of N2O (Zhu et al., 2008, 2013). During denitrification, the lighter isotopes (14N and 16O isotopes) are preferentially used by microorganisms, leaving the residual NO3− enriched in 15N and

18O.

Thus, the δ18O-NO3– value of soil was significantly higher in the active

penguin colony. Second, the contribution of NO3− from atmospheric deposition in M soil increased because the oxygen consumption of soil organic matter increased at the active penguin colony. Thus, the oxygen supply for nitrification decreased, leading to the decrease of NO3− from nitrification, and the proportion of N derived from atmospheric deposition increased. In this case, the nitrate is more enriched in 18O. The δ18O-NO3– in total suspended particulates (TSP) in East Antarctica is about 100‰ in 20

late winter and early spring (Frey et al., 2009). According to a widely used global precipitation oxygen isotope model (Bowen and Wilkinson, 2002), the known parameters with latitude of ~74°S and elevation of approximately 0, then the calculated δ18O value of atmospheric precipitation on Inexpressible Island is approximately −19.82‰. In addition, δ18O of O2 from atmosphere is constant with the value of 23.88‰ (Barkan and Luz, 2005). During the nitrification process, NH4+ is converted to NO2− by reactions with H2O and O2, and then converted to NO3− by H2O (Casciotti, 2016). Thus, two oxygen atoms are from H2O, and the other from O2. Therefore, the δ18O value in NO3− from nitrification should be about (−19.82 × 2 + 23.88 × 1)/3‰, i.e., approximately −5.25‰. Assuming there are two NO3– sources on Inexpressible Island (nitrification and atmospheric deposition), a two-end member linear mixing model was applied to assess the contributing proportion of NO3– from nitrification (f) and atmospheric deposition (1−f). Thus, δ18O-NO3−soil = −5.25‰ × f + 100‰ × (1−f), the calculated f-value is about 82% in M soils, and the f value is close to 100% in the F and A soils. All the above calculated results imply that nitrification is the dominant pathway of NO3− production in the F and A soils on Inexpressible Island, while for M soils, in addition to the contribution of nitrification, there was also a small fraction of NO3– from atmospheric deposition.

4.2. Guano input and moss N utilization

The higher N concentrations and δ15N values of moss in guano-impacted areas 21

(Figs. 3, 4), and positive correlations between soil P and soil δ15N-TN values (r=0.840, n=31, P<0.01) and between δ15N-TN values of moss and moss-growth soil (r=0.853, n=31, P<0.01), all indicate that the penguin-derived N input to the soil may have caused significant influence on the moss N utilization strategies. Although many studies have reported that mosses are excellent scavengers of atmospheric N, soil N may be also as important as a N source to moss (Ayres et al., 2006; Liu et al., 2013a, 2018). From the perspective of inorganic N isotopes, we found that in guano-free areas there were no significant differences between moss δ15N-TN values and soil δ15N-NH4+ and δ15N-NO3– values (Fig. 4). We assumed that there was little N isotope fractionation when moss uptake inorganic N in these areas. However, there were no significant correlations between moss δ15N-TN values and inorganic N isotopes, perhaps attributed to the moss also uptaking dissolved organic N (DON) since the amount of organic N is the most abundant in moss-growth soil, regardless if impacted by guano or not. Previous research has also shown that moss can utilize a significant proportion of free amino acids from the soil where inorganic N sources are scarce (Krab et al., 2008; Wiedermann et al., 2009). However, amino acids only account for a small proportion of DON and its bioavailable fraction is expected to be larger. Here, based on the organic N as the main form of soil TN, we speculate that soil DON may also be used as an important N source for the growth of moss in Victoria Land, with the exception of natural atmospheric nitrogen deposition. To compare the 22

moss N uptake strategy in guano-free and guano-impacted soils, we assumed that there are three dissolved N sources (NH4+, NO3–, DON) in the soil, and N uptake by moss has no substantial 15N fraction (Liu et al., 2013a). Since the δ15N values of DON have not been determined, we roughly replaced it with δ15N values of total organic N (ON). First, based on the concentrations of soil inorganic N and TN, we calculated the proportion of soil organic N to TN as follows: 𝑓𝑂𝑁 = 1 ― (

𝐶𝑁𝐻4 + 𝐶𝑇𝑁

+

𝐶𝑁𝑂3 ― 𝐶𝑇𝑁

)

(1)

Then, we obtained the δ15N values of ON according to the mixing model: 𝛿15𝑁­𝑇𝑁𝑠𝑜𝑖𝑙 = 𝛿15𝑁­𝑁𝐻4+ 𝑠𝑜𝑖𝑙 × 𝑓NH4+ + 𝛿15𝑁­𝑁𝑂3― 𝑠𝑜𝑖𝑙 × 𝑓𝑁𝑂3― + 𝛿15𝑁­𝑂𝑁𝑠𝑜𝑖𝑙 × 𝑓𝑂𝑁

(2)

where f is the proportion of each N form to TN. Finally, if we only consider the contribution of soil nitrogen to moss uptake, the proportional contributions (f) of each N form are calculable based on the IsoSource model (Phillips and Gregg, 2003). Here, the value of δ15N-DON was replaced with δ15N-ON: 𝛿15𝑁­𝑇𝑁𝑚𝑜𝑠𝑠 = 𝛿15𝑁­𝑁𝐻4+ 𝑠𝑜𝑖𝑙 × 𝑓1 + 𝛿15𝑁­𝑁𝑂3― 𝑠𝑜𝑖𝑙 × 𝑓2 + 𝛿15𝑁­𝐷𝑂𝑁𝑠𝑜𝑖𝑙 × 𝑓3

(3)

where f1+f2+f3 =1. The result demonstrates that in the guano-free area, the mean proportional contributions of soil NH4+, NO3–, and DON were 21±17%, 44±24% and 35±20%, respectively. While in the guano-impacted area, the mean proportional contributions of these three N forms were 24±20%, 20±13% and 56±15%, respectively (Fig. 6). It was obvious that the proportional contribution of DON to 23

moss N significantly increased in the ornithogenic soil. Therefore, given mosses utilize the soil N, we conclude that in the absence of guano, soil inorganic N might be the main source to moss growth, and the contribution of nitrate in the soil cannot be ignored. Indeed, previous research has shown that soil NO3−-N is also an important N source for Arctic tundra plants (Liu et al., 2018). Although the NO3−-N concentrations were typically low in the soil, plants took up NO3−-N and NH4+-N at similar rates or even higher for the former (Liu et al., 2018). In the guano-impacted area, soil DON is likely to become an important source to moss. Previous study has shown that DON was an important N source for moss N utilization, with a mean contribution of 38% (Liu et al., 2013a). Although the contribution of atmospheric deposition was not considered in our model, the results still demonstrated that the contribution of soil DON should not be neglected when considering moss N utilization in extremely cold east Antarctic terrestrial ecosystems, especially in and near penguin colonies.

4.3. Gradient change on δ15N values of aquatic microbial mats

In addition to the significant impact on the soil and plants of terrestrial ecosystem function, penguin activities had a similar effect on microbial mats in ponds and lakes. Uric acid hydrolysis can lead to a temporary increase in pH, a condition that favors the formation of ammonia. Ammonia is easily volatilized to the atmosphere and a large kinetic isotope fractionation accompanies it, resulting in ammonia that is strongly 15N depleted while the remaining ammonium is 15N enriched (Heaton, 1986). 24

However, the recent result of Crittenden et al. (2015) showed that ammonia emitted from a penguin colony at Cape Hallett had positive δ15N values (6.94±2.26 ‰), reflecting

15N

enriched in the penguin guano and ornithogenic soils. Moreover,

Crittenden et al. (2015) reported that the lichen δ15N values were strongly positively correlated to the proximity to the active penguin colony, and high δ15N values were present in the lichens closer to the colony because of large contribution of penguin-derived NH3. Thus, we speculate that the microbial mats assimilating 15N-enriched

N near the penguin colonies have positive δ15N values, while the

microbial mats assimilating

15N-depleted

abiogenic N at upland sites have negative

δ15N values. The δ15N values in the microbial mats from the IIL9 pond at the middle distance from the penguin colony were ~ −0.5‰, and this was apparently due to the contribution of N2-fixing cyanobacteria (Wada et al., 2012), implying the significant decrease of guano-derived N influence. The aquatic microbial mats in the fellfield sites far from the active penguin colony had more negative δ15N values from −12.9‰ to −10.7‰, indicating that these aquatic microbial mats were significantly less affected by penguin guano, and more affected by the atmospheric N precipitation which usually had negative δ15N values. Some studies have shown that the epibenthic algae collected from the Dry Valleys, South Victoria Land, had very low δ15N values up to -50‰ under conditions of high nitrate concentration and low light intensity, and this was explained by the large isotope fractionation associated with nitrate assimilation by algae or microbial mats in the ponds, while the precipitated source 25

nitrate also had low nitrogen isotope values (~ −20‰) (Wada et al., 1981, 1984, 2012). Since there were no accurate δ15N values of gaseous ammonia and precipitated source nitrate in the study area, the real reasons for the relatively negative δ15N values of microbial mats in the ponds and lakes far from the active penguin colony warrant further analysis. Nevertheless, it is obvious that distance from the penguin colony is an important factor influencing the microbial mat N utilization (Fig. S1, SI Appendix). Our results are consistent with previous studies in other Antarctic and subantarctic regions, where the N concentrations and δ15N values of plants decreased with increasing distance from penguin colonies (Erskine et al., 1998; Crittenden et al., 2015; Bokhorst and Convey, 2016). Also, research at the subantarctic Macquarie Island showed that the mean δ15N values of plants growing near animal nests was 12.9‰, while the mean values of plants at upland plateau sites were below −5‰ (Erskine et al., 1998). Similar to the terrestrial moss N utilization strategies described above, the result of aquatic microbial mat N isotope signatures suggests that the nitrogen derived from penguin colonies can significantly affect the N utilization strategies of aquatic microbial mats at different distances from the penguin colony.

4.4. Dynamic model of penguin-derived N

Based on data comparison of nitrogen species and their isotope compositions in soil and plants from the guano-free and guano-impacted areas, we have presented a preliminary dynamic model of penguin-derived N in the terrestrial and aquatic 26

ecosystems near penguin colonies (Fig. 7). The penguin colony serves as an important N source with guano increasing the N abundance in the surrounding environment. Organic N is the main species of penguin-derived N, and it can rapidly mineralize to highly bioavailable inorganic forms, such as NH4+ and NO3−, increasing the concentrations of soil inorganic N, and causing the soil to be

15N-enriched

near the

penguin colonies. The mosses affected by guano were more enriched in δ15N than those in guano-free areas where soil inorganic N might not be ignored for moss N uptake, with the exception of natural atmospheric deposition. With the increasing effect of guano-derived N, the soil DON may become an important N source to the mosses in the guano-impacted area. Apart from the direct input to soil, volatile ammonia during degradation of penguin guano is also a key process that controls the distribution of N sources. Preliminary estimates of NH3 emission from seabirds suggest that they were a significant natural source (Sutton et al., 2000; Wilson et al., 2004). Although far away from penguin colonies, the bare soils are still affected by the volatile ammonia of penguin guano. Given the significant impact of penguins on Antarctic terrestrial and aquatic ecosystems, changes in penguin distribution driven by climate change and human activities will have a major impact on local terrestrial and aquatic ecosystems. Future research should consider the loss of penguin-derived N such as leaching, runoff, and denitrification, because the loss of N is also a key process of the N cycle, to quantify the conceptual model and intuitively depict the impact of seabird activity on the N cycle at penguin colonies in Antarctica. 27

5. Conclusions

We conducted a comprehensive study on the impact of penguins on N dynamics in Victoria Land, especially at Inexpressible Island, and preliminarily demonstrate a conceptual model. Both N concentrations and isotope ratios indicated that penguins have a strong influence on the N cycle in their surrounding ecosystem in Antarctica. Penguin guano not only acts as an important N source for the island, but also plays a key role in plant N utilization. The guano could cause a large impact zone outside the penguin colonies. In addition, penguin-derived N affects N utilization strategies in moss growing nearby. Mosses affected by guano had higher N isotope values, and the soil DON related to the guano input may become an important nitrogen source for moss uptake. For aquatic microbial mats, the samples closer to penguin colony were 15N-enriched,

while 15N-depleted at upland sites. This study emphasizes a key role of

penguins in the N cycle in remote ecosystems in Antarctica.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant numbers. 41576183 and 41776188) and by NSF Grant ANT-1443585 to S. Emslie. We thank the Chinese Arctic and Antarctic Administration of the State 28

Oceanic Administration for project support. We also thank the United States Antarctic Program (USAP), Antarctic Support Contract and the Italian Mario Zucchelli Station for logistical support. R. Murray and A. McKenzie provided valuable assistance in the field. We are especially grateful to Linlin Zheng and Yun Wei for their help in stable isotope analyses. Dr. Pengcheng Wu and Jian Yang are acknowledged for their help in moss species identification. Dr. Yaguang Nie is thanked for his valuable help to prepare this manuscript. We thank Dr. Songtao Ai for his help to obtain the data of relative distance and relative elevation of microbial mat sampling sites. We acknowledge four anonymous reviewers for their valuable suggestions to greatly improve our paper.

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42

Figures Fig. 1. Map of the study area. (A) the location of Cape Irizar, Inexpressible Island, North Adélie Cove, Campo Icarus and Cape Hallett in Victoria Land, Ross Sea, Antarctica. (B) Inexpressible Island. (C) Microbial mats (red tag) and some of the moss (green circle) and bare soil sampling sites (stars) at Inexpressible Island; the blue pattern represents ponds and lakes.

43

Fig. 2. Soil P, TOC, and TN concentrations (A), δ15N-TN, δ15N-NO3−, and δ15N-NH4+ values (B), soil NH4+-N and NO3–-N concentrations (C), and δ18O-NO3− values (D) from different sampling sites: F (far away from colony), n=3; M (modern

colony),

n=7; and A (abandoned colony ), n=3. For each bar, different letters indicate significant differences (t-test, P<0.05) among soil types.

44

Fig. 3. Moss-growth soil P (A), TOC (B) and TN (C) concentrations, as well as moss bulk C (D) and N (E) concentrations, with or without guano influence at Cape Irizar, Inexpressible Island, North Adélie Cove, Campo Icarus and Cape Hallett.

45

46

Fig. 4. The nitrogen isotope composition (δ15N) of moss N, inorganic N (NH4+-N, NO3–-N) and TN in moss-growth soils with (n=5) and without (n=14) guano influence at Cape Irizar, Inexpressible Island, North Adélie Cove, Campo Icarus and Cape Hallett.

47

Fig. 5. TN concentrations and δ15N-TN values of microbial mats in the ponds and lakes at Inexpressible Island.

48

Fig. 6. Proportional contributions (mean ± SD) of soil NH4+-N, NO3−-N and DON to moss N, with and without guano influence.

49

Fig. 7. Preliminary dynamic model of penguin-derived N in the Antarctic ecosystem near penguin colonies.

50

Tables Table 1 Correlation analysis of bare soil P, TOC, N, NH4+ and NO3− concentrations, as well as δ15N-TN, δ15N-NH4+, δ15N-NO3− , and δ18O-NO3− values from F (far away from colony), M (modern colony) and A (abandoned colony) sites at Inexpressible Island (n=13).

P TOC P

TOC

TN

NH4+-N

TN

NH4+- NO3-- δ15N-

δ15N-N

δ15N-N

δ18O-N

N

1 0.939 0.860 0.744 **

N

TN

H4+

O3−

O3−

0.635

0.530

0.369

0.102

0.670*

0.456

0.488

-0.005

0.778**

0.266

0.390

-0.133

0.817**

0.298

0.299

-0.116

0.687**

0.231

0.418

-0.153

0.571*

1

0.445

0.387

0.410

1

0.287

0.704**

1

0.115

**

**

*

1

0.903 0.692

0.765

**

**

**

1

0.891

0.717

**

**

1

0.607 *

NO3--N

1

δ15N-T N δ15N-N H4+ δ15N-N 51

O3− δ18O-N

1

O3− * The correlation was significant above 0.05. ** The correlation was significant above 0.01.

52

53

Highlights: 1. δ15N of TN and inorganic N (NH4+ and NO3−) in soil, moss and aquatic microbial mat were investigated. 2. δ15N values of soil far outside the penguin colonies resembled those in penguin colonies. 3. The moss impacted by guano was more enriched in δ15N than in guano-free areas. 4. Aquatic microbial mat near penguin colonies was 15N-enriched, while 15N-depleted at upland sites.

54