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. 2016 Apr 16;117(7):1153–1161. doi: 10.1093/aob/mcw058

Sensitivity of the xerophytic moss Syntrichia caninervis to prolonged simulated nitrogen deposition

Yuanming Zhang 1,*, Xiaobing Zhou 1, Benfeng Yin 1,2, Alison Downing 3
PMCID: PMC4904175  PMID: 27085182

Abstract

Background and Aims Biological soil crusts, comprising assemblages of cyanobacteria, fungi, lichens and mosses, are common in dryland areas and are important elements in these ecosystems. Increasing N deposition has led to great changes in community structure and function in desert ecosystems worldwide. However, it is unclear how moss crusts respond to increased atmospheric N deposition, especially in terms of growth and physiological parameters. The aim of this study was to understand how Syntrichia caninervis, a dominant species in moss crusts in many northern hemisphere desert ecosystems, responds to added N.

Methods The population and shoot growth, and physiological responses of S. caninervis to six different doses of simulated N deposition (0, 0·3, 0·5, 1·0, 1·5 and 3·0 g N m–2 year–1) were studied over a 3 year period.

Key Results Low amounts of added N increased shoot length and leaf size, whereas high doses reduced almost all growth parameters. Moss shoot density increased, but population biomass decreased with high N. Low N augmented chlorophyll b, total chlorophyll content and soluble protein concentrations, but not chlorophyll a or chlorophyll fluorescence. High N was detrimental to all these indices. Soluble sugar concentration declined with increased N, but proline concentration was not affected significantly. Antioxidant enzyme activities generally decreased with low N additions and increased with high doses of simulated N deposition.

Conclusions Low amounts of added N (0–0·5 g N m–2 year–1) may enhance moss growth and vitality, while higher amounts have detrimental effects.

Keywords: Antioxidant enzyme, chlorophyll, fluorescence, nitrogen deposition, osmotic substance, Syntrichia caninervis

INTRODUCTION

Biological soil crusts (BSCs), complex communities of cyanobacteria, fungi, lichens and mosses, are a major component of the soil surface in arid areas (Evans and Johansen, 1999). In some arid lands, BSC cover may exceed 70 % of the land surface (Belnap, 1995). BSCs may affect important ecological processes in desert ecosystems, such as sand surface stability (Eldridge and Greene, 1994; Hu et al., 2002; Zhang et al., 2006), the hydrological cycle, plant growth and community structure (Evans and Johansen, 1999; Belnap, 2006; Liu et al., 2006; Li et al., 2011), and nitrogen (N) sources (Belnap, 2002; Zaady, 2005; Stewart et al., 2011).

Moss crusts generally develop at advanced stages of crust succession and often form a mosaic in lichen or cyanobacterial crusts (Lan et al., 2012). Mosses are important floristic constituents of many dryland ecosystems and play an important role in the carbon balance of BSCs (Coe et al., 2012b). Being poikilohydric, mosses maintain the moisture content of their cells at equilibrium with the surrounding atmosphere. Mosses also exhibit several morphological and structural adaptations to dry environmental conditions throughout their life cycles (Zheng et al., 2011) and have the potential to resume photosynthesis rapidly after wetting, even in low temperatures and in low light (Proctor and Smirnoff, 2000; J. Zhang et al., 2011), and thus can survive extremely hot summers, bitterly cold winters and severe, protracted drought (Stark et al., 2012).

Because mosses acquire most of their nutrients from the atmosphere, they are extremely useful as bioindicators (Ochoa-Hueso and Manrique, 2013; Ochoa-Hueso et al., 2014) and should be included in studies of the impacts of environmental changes. There are already strong indications that rapid climate changes and anthropogenic disturbance greatly influence moss crusts (Belnap and Eldridge, 2003). For example, changes in timing, seasonality, frequency and intensity of rainfall events have been shown to alter the above-ground biomass and surface cover of the dominant moss Syntrichia caninervis (Coe et al., 2012b; Kidron et al., 2012; Reed et al., 2012) and, when grown in elevated CO2, biocrust mosses showed a decreased chlorophyll content and increased carbon uptake, but N content and photosynthetic performance remained unchanged (Coe et al., 2012a).

Aside from elevated CO2, increasing N deposition because of greater emission of N gas and particulates from continuously encroaching agricultural and industrial developments (Vitousek et al., 1997; Fenn et al., 2003; Bobbink et al., 2010) is also posing major threats to desert ecosystems. Since N is limited in desert soils, any increase in N deposition has the potential to modify plant growth and associated community structure greatly (Throop, 2005; Clark and Tilman, 2008). BSCs have already suffered negative impacts (Belnap et al., 2008; Ochoa-Hueso et al., 2014) including the invasion of annual exotic grass speciess (Belnap et al., 2006). Previous studies of the effects of added N on mosses have mainly focused on forest ecosystems where the responses of mosses to N addition vary among species, study sites and N concentrations (Jones et al., 2002; Pearce et al., 2003). In glasshouse trials modelling semi-arid Mediterranean ecosystems, addition of N, irrespective of quantity, resulted in increased moss cover, whereas under field conditions, N deposition affected moss physiology but not the extent of cover (Ochoa-Hueso and Manrique, 2013). The few comparable studies carried out to date on desert moss crusts indicate that low amounts of added N promote shoot burning (chlorosis) and negatively affect regeneration responses, whereas high amounts may be toxic (Soares and Pearson, 1997; Pearce et al., 2003) and inhibit the resumption of apical meristematic growth after desiccation (Stark et al., 2011). We therefore undertook a detailed 3 year study of the effects of added N on a range of growth (population density, biomass and leaf dimensions) and physiological parameters (concentrations of osmotic adjustment substances, and antioxidant enzyme activities) using S. caninervis, a widespread and often dominant moss species in arid northern hemisphere ecosystems.

The objectives were to (1) evaluate the growth and physiological characteristics of S. caninervis under different doses of added N and (2) test if there is a critical N concentration for optimal moss growth. Given the limitation of N in desert ecosystems, we hypothesized that (a) growth- and photosynthesis-related physiological activity of S. caninervis would be stimulated by the addition of small amounts of N, but would be suppressed by higher N concentrations (Pearce et al., 2003); and (b) the concentration of osmotic adjustment substances and antioxidant enzyme would show opposite trends to growth because more resources would be allocated to synthesis and maintenance of defensive compounds (Bazzaz et al., 1987).

Our study site was the Gurbantunggut Desert, the second largest desert in China, which, in recent years, has been experiencing increased N deposition (Jia et al., 2014). In desert areas close to the city of Urumqi, the rate of dry N deposition may reach 2·9 g N m−2 year−1 (Zhang et al., 2011), which has had significant effects on biomass and regeneration of moss crusts in other desert and semi-arid areas (Stark et al., 2011; Ochoa-Hueso and Manrique, 2013). However, this provides little or no clue as to how lower rates of N deposition, <1 g N m–2 year–1, further away from the city (Li et al., 2012) might affect the moss.

MATERIALS AND METHODS

Study site

The study site (44·62'N, 88·26'E, 570 m a.s.l.) is located in the centre of the Gurbantunggut Desert, north-western China. The desert has a mean annual temperature of 8 °C and mean annual precipitation of 70–150 mm, of which half (47·6 %) falls between April and July. In winter, about 20 cm of snow covers the sand surface. In spring, the melting snow together with increasing rainfall leads to abundant soil moisture which is favourable for the emergence of many annual plant species: Erodium oxyrrhynchum Bieb., Ceratocarpus arenarius L., Haloxylon ammodendron (C. A. Mey) Bunge ex Fenzl and H. persicum Bunge ex Boiss. & Buhse are the dominant desert shrubs. The desert is characterized by massive fixed and semi-fixed sand dunes, with roughly 40 % coverage by different types of BSCs, distributed from the dune crest to the inter-dune area (Zhang et al., 2010). The peak BSC growing period is in the cooler and wetter months of the year, when dew, fog and rainfall events are more frequent. Syntrichia caninervis is the dominant moss species in desert BSCs.

Field methods

In October 2010, ten replicated blocks, each consisting of six 2 × 2 m plots (2 m intervals), were set out at the study site. All plots were located in flat inter-dune areas, with well-developed lichen and moss BSCs. Vascular plant composition and density, and soil physiochemical characteristics were similar in all plots.

Five N addition treatments and a control (no added N) were randomly applied to the six plots of each block. The six treatments, each with ten replicates, were 0, 0·3, 0·5, 1·0, 1·5 and 3·0 g N m–2 year–1, henceforth designated N0, N0·3, N0·5, N1, N1·5 and N3, respectively. The N addition rates were within the range of natural N deposition rates at sites near the desert margin. The treatments were applied in equal amounts in October before snowfall and in March after snow thaw from October 2010 to October 2013. We selected these times because field cultivation and fertilizer application in farmland near the desert peak in March and October, with the strong likelihood of increased N emission and deposition onto desert soils. Each N application consisted of 2:1 NH4:NO3 (NH4NO3 and NH4Cl), which approximates the composition of N deposition recorded for nearby oases (Zhang et al., 2008), thus reproducing the impact of N deposition from nearby oases to desert ecosystems.

The N for each plot was dissolved in 10 L of water and sprayed evenly across the surface. In March, at the time of N application, the plots were moist from snow melt. In October, the N additions were applied either after small rainfall events or in the early morning when BSCs were wet with dew, in order to avoid additional stress (rapid desiccation) by the N application to the surface of the moss BSCs.

Fluorescence measurements

Four blocks were randomly selected for the fluorescence measurements and subsequent sample collection. In March 2014, at 2 d after the end of snow melt when the moss shoots were hydrated, and 1 d before the next N application, we selected plots from the four blocks in which to measure chlorophyll fluorescence of S. caninervis in situ using a portable pulse amplitude fluorometer (PAM 2500, Walz, Inc., Effeltrich, Germany). The mosses remained hydrated during the entire period of fluorescence measurement. Actual photochemical efficiency Y(II) of mosses was measured under ambient light. For measurement of maximum quantum yield (Fv/Fm), the saturation pulse method was used. The moss population sample used for Fv/Fm measurements was monitored in the dark in a box covered for >30 min. Fo was obtained on excitation with a weak measuring beam, and maximal fluorescence yield Fm measured following a 0·8 s saturating pulse (15 000 μmol m–2 s–1) to close all reaction centres. The Fv/Fm was calculated as (Fm – Fo)/Fm. Four points of moss crusts were selected in each plot. Y(II) and Fv/Fm at four points were averaged to represent one plot.

Sample collection

After the fluorescence measurements, samples of soil and of S. caninervis were collected from the above four blocks. For soil samples, three cores, each 5 cm in diameter and 0–5 cm deep, were collected from each treatment plot within each of the four blocks and mixed to form one composite sample. The samples were air-dried for physicochemical analysis. Soil pH and electrical conductivity (EC) were measured in a 1:5 mixture of soil and deionized water with pH and EC meters, respectively. Soil organic matter (OM) was determined by the K2Cr2O7 method (Walkley–Black); total N by the CuSO4-Se powder diffusion method; total phosphorus (P) by the NaOH fusion–Mo TeSc colorimetry method; and available N by the alkali hydrolyzation–diffusion method (Chen et al., 2007).

Syntrichia caninervis was collected in PVC tubes (11 cm diameter, 5 cm depth), placed in a portable ice-box and immediately transferred to the laboratory. The contents of some tubes were used to measure morphology and biomass; the other tubes were used to determine physiological characteristics, such as soluble sugar, proline, soluble protein and antioxidant enzyme activity. The photosynthetically active leaves and young stems were promptly removed using a sharp blade, and sand grains were carefully detached using mesh. Leaves and stems were wrapped in aluminium foil, frozen in liquid N, and stored at − 80 °C for physiological analysis.

Moss population density and biomass

A 1 × 1 cm frame was used to select moss samples in the PVC tube for shoot density and biomass measurements. Individual moss plants were separated and washed in a Petri dish to remove sand grains. We use ‘individual’ to refer to one whole shoot, comprising both above- and below-ground components. The number of individuals was recorded. The 1 cm2 moss samples were oven dried at 65 °C to constant weight and the dry biomass recorded to 0·0001 g accuracy.

Individual size and leaf growth characteristics

Biomass accumulation of individuals was calculated based on moss density and population biomass. Twenty individuals from different N treatments were randomly selected for morphological measurements. Individuals were placed in a Petri dish and distilled water added to ensure the plants remained hydrated and alive. The Petri dish was moved to a stereomicroscope to capture images of the shoot length using the same scale. A short needle was used as a reference measure. Shoot length was measured from the apex of the stem to the starting point of the below-ground section. For measurement of leaf length and width, and hairpoint length, leaves were carefully removed from the same moistened shoots. Each leaf was photographed under a light microscope (×10 objective). All measurements were recorded from the image using Image J software (National Institutes of Health, Bethesda, MD, USA).

Chlorophyll measurement

For chlorophyll determination, 0·15 g of fresh moss shoots were weighed and ground in liquid N and CaCO3 with a mortar and pestle. The pigments were extracted in 95 % (v/v) chilled ethanol in the dark and incubated overnight at 4 °C. The samples were centrifuged (4000 g, 10 min), after which the absorbance of the filtered extract was measured at 665, 649 and 470 nm. The chlorophyll concentration was calculated in accordance with the Lichtenthaler formula on a dry weight basis (Lichtenthaler and Wellburn, 1983).

Measurements of soluble sugar, proline, soluble protein concentrations and antioxidant enzyme activity

Soluble sugar was determined based on the method of Lassouane et al. (2013). Fresh tissue (leaf and stem together, 0·15 g) was ground to a fine powder in liquid N and mixed with 7 mL of 70 % ethanol (v/v) for 5 min on ice. The sample was centrifuged at 8000 g for 10 min at 4 °C. A sub-sample (200 mL) of the extract was mixed with 1 mL of anthrone and heated in a boiling water bath for 10 min. The absorbance was recorded at 625 nm. Glucose was used as the standard to calculate the soluble sugar concentration.

Proline concentration was measured in accordance with the methods of Bates et al. (1973) and Monreal et al. (2007), with some modifications. Specifically, 0·15 g of fresh tissue was ground in 5 mL of 3 % sulphosalicylic acid, and extracted in boiling water for 20 min. After centrifuging, 2 mL of supernatant was mixed with acetic acid and acid ninhydrin, and incubated in an oven at 100 °C for 40 min. The mixture was cooled at ambient temperature, then 5 mL of toluene was added and the absorbance was recorded at 520 nm.

To determine the soluble protein concentration, 0·15 g of fresh tissue was ground in deionized water at 4 °C, then centrifuged at 8000 g for 30 min. A 5 mL aliquot of Coomassie brilliant blue was added to 200 μL of protein extracts. Absorbance of soluble protein was read at 595 nm. Bovine serum albumin was used as the standard (Gonzalez and Pignata, 1994). The above three osmotic adjustment substances were expressed as dry weights.

Peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activities were determined using the methods of Choudhury and Panda (2005), Sun et al. (2009) and Wu et al. (2012). Specifically, 9 mL of 0·2 m phosphate buffer (pH 7·0) was added to 0·15 g of ground fresh tissue, under ice-cold conditions. The mixture was centrifuged at 3500 g for 10 min. The supernatant was used for the measurement of POD, SOD and CAT activity. For POD, the H2O2 reaction mixture was added to the supernatant and the absorbance recorded at 420 nm. The SOD activity was calculated after recording the absorbance at 550 nm. One unit of SOD refers to the amount of enzyme that inhibits the reduction of nitroblue tetrazolium by 50 %. The CAT activity was estimated based on disappearance of H2O2, and the absorbance was measured at 405 nm. Activities of POD, SOD and CAT were calculated based on soluble protein.

Statistical analysis

The average moss density, size, leaf length and width, and hairpoint length were calculated for each plot. We employed a one-way analysis of variance (ANOVA) to assess the significance of differences among treatments, and the least significant difference (LSD) was performed to determine whether differences were statistically significant. All the data were tested for homoscedasticity and normality before analysis. All comparisons were performed using SAS 6.1 software (SAS Institute, Inc., Cary, NC, USA).

RESULTS

Soil physicochemical characteristics

The addition of nitrogen reduced soil pH, with significant decreases recorded for N3 treatments (Table 1, P < 0·05). However, N3 addition significantly increased EC compared with the control (N0). Organic matter, total N and available N were not significantly affected by the N treatments (P > 0·05).

Table 1.

Physicochemical characteristics of the soil in response to different N application rates

N treatments pH (1:5 w/v) EC (μs cm−1) OM (g kg−1) Total N (g kg−1) Total P (g kg−1) Available N (mg kg−1)
N0 7·7 ± 0·1a 78·67 ± 4·6b 3·01 ± 0·21a 0·19 ± 0·01a 0·35 ± 0a 16·87 ± 0·91a
N0·3 7·6 ± 0a 80·67 ± 4·48ab 3·13 ± 0·65a 0·16 ± 0·05a 0·35 ± 0·02a 18·85 ± 1·79a
N0·5 7·6 ± 0·1ab 79 ± 3·21b 2·97 ± 0·49a 0·17 ± 0·04a 0·34 ± 0·01a 17·77 ± 2·58a
N1 7·6 ± 0ab 82 ± 4·73ab 2·5 ± 0·27a 0·19 ± 0·01a 0·34 ± 0·01a 14·88 ± 2·42a
N1·5 7·6 ± 0ab 93·33 ± 9·35ab 2·28 ± 0·25a 0·16 ± 0·04a 0·35 ± 0·01a 15·51 ± 2·91a
N3 7·4 ± 0·1b 97 ± 5·03a 2·39 ± 0·14a 0·16 ± 0·02a 0·33 ± 0·01a 18·28 ± 1·7a
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EC, electrical conductivity; OM, organic matter.

Moss population density and biomass accumulation

No significant differences in population density were observed between the controls and treatments with lower quantities of added N (N0·3 and N0·5) (P > 0·05; Fig. 1). Higher quantities of added N (N1–N3) significantly increased the population density (number of shoots per 1 cm2). No significant differences were observed among high N (N1–N3) treatments (P > 0·05). There was no increasing trend in population density corresponding to increased amounts of added N.

Fig. 1.

Fig. 1.

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Population density (number of individuals per 1 cm2) of Syntrichia caninervis in response to different levels of N addition (mean ± s.e., n = 4). Different letters above a bar indicate a significant difference (P < 0·05).

In contrast, accumulation of population total biomass showed a gradually decreasing tendency as the rate of N addition increased from N0·3 to N1·5, though differences were not significant among N0–N1·5 treatments (P > 0·05; Fig. 2). However, population biomass decreased significantly at the highest N addition treatment (N3; P < 0·05).

Fig. 2.

Fig. 2.

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Dry biomass accumulation by 1 cm2 Syntrichia caninervis populations in response to different levels of N addition (mean ± s.e., n = 4). Different letters above a bar indicate a significant difference (P < 0·05).

Growth response of individual plants to N addition

Figure 3 shows the differences in growth characteristics of individual plants and leaf dimensions under different N treatments. Added N significantly influenced individual plant biomass, shoot lengths and leaf dimensions (Fig. 4A–C; P < 0·05). Individual plant biomass was highest in the N0·3 treatment and then decreased gradually with increasing amounts of N (Fig. 4A).

Fig. 3.

Fig. 3.

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Representative shoots (upper images) and leaves (lower images) of Syntrichia caninervis from the N addition treatments illustrate the different shoot lengths and leaf length.

Fig. 4.

Fig. 4.

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(A) Individual plant biomass (mean ± s.e., n = 4); (B) shoot length (mean ± s.e., n = 20); (C) leaf length, leaf width and hairpoint length (mean ± s.e., n = 15) of Syntrichia caninervis in response to different levels of N addition. Different letters above a bar indicate a significant difference (P < 0·05).

The highest amounts of added N (N1 –N3) reduced individual biomass and shoot lengths (Fig. 4A, B). Low amounts of N addition (N0·3–N0·5) positively affected leaf length and width, and hairpoint length (Fig. 4C). The highest rate of N addition (N3), however, reduced these leaf dimension parameters. The results demonstrated that high rates of N addition negatively impacted on the growth of S. caninervis.

Chlorophyll fluorescence parameters

Chlorophyll concentration increased at low amounts of added N (N0·3) then gradually decreased with increasing N addition (Fig. 5). However, chlorophyll a concentrations were not significantly affected by added N when compared with the control (N0) (P > 0·05). The values of both Y(II) and Fv/Fm showed a gradual downward trends with increasing amounts of added N, although, with the exception of the N3 treatment, the differences were not significant (Fig. 6; P < 0·05).

Fig. 5.

Fig. 5.

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Concentration of chlorophyll a, b and a + b of Syntrichia caninervis in response to different levels of N addition (mean ± s.e., n = 4). Different lower case letters (chlorophyll a and b) and upper case letters (chlorophyll a + b) above a bar indicate a significant difference (P < 0·05).

Fig. 6.

Fig. 6.

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Actual photochemical efficiency [Y(II)] and maximum quantum yield (Fv/Fm) of Syntrichia caninervis in response to different levels of N addition (mean ± s.e., n = 4). Different letters above a bar indicate a significant difference (P < 0·05).

Concentration of osmotic adjustment substances and antioxidant enzyme activities

With an increasing amount of added N, soluble sugar, soluble protein and proline concentrations showed differing trends (Fig. 7). Soluble sugar increased slightly at N0·3 (P > 0·05) but then gradually decreased with increasing amounts of N (P < 0·05). Soluble protein increased from N0·3 to N1 (P > 0·05), then decreased at N1·5 and N3. No changes in proline concentrations were observed (P > 0·05).

Fig. 7.

Fig. 7.

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Concentration of osmotic adjustment constituents of Syntrichia caninervis in response to different levels of N addition (mean ± s.e., n = 4). Different letters above a bar indicate a significant difference (P < 0·05).

Similar trends for changes in activities of SOD, POD and CAT were observed with increasing N additions from N0 to N3 (Fig. 8). However, although the mean activity of the three enzymes varied greatly, significant differences were only observed for POD.

Fig. 8.

Fig. 8.

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Antioxidant enzymes peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) of Syntrichia caninervis in response to different levels of N addition (mean ± s.e., n = 4). Different letters above a bar indicate a significant difference (P < 0·05).

DISCUSSION

Non-linear responses of moss growth to N enhancement

Low amounts of added N clearly increased individual plant biomass, shoot size and leaf size in the desert moss Syntrichia caninervis, whereas more added N decreased these parameters. These results are consistent with the initial hypothesis that moss growth is stimulated by low N addition, and suppressed by larger amounts of added N (for example Jones et al., 2002; Jägerbrand et al., 2003; Pearce et al., 2003). Whereas the addition of > 1 g N m–2 year–1 was clearly detrimental to S. canivervis from the Gurbantunggut Desert, the same species from the Mojave Desert in western North America showed no significant shoot biomass differences for various N treatments (0–4 g N m–2 year–1) (Stark et al., 2011). We attribute this difference to the greater number of N application events in the Gurbangunggut Desert each year (twice vs. once), or the differet rainfall patterns in the two desert.

Images of individual moss shoots from N1·5 and N3 plots (Fig. 3) revealed shoot burning (chlorosis) due to the large amounts of added N, resulting in much smaller shoots than the controls. This result also differs from the findings Stark et al. (2011), who recorded significantly higher shoot chlorosis with low amounts of added N (1 g) but not for higher amounts.

The positive benefits of the application of small amounts of added N (0·3–0·5 g N m–2 year–1) observed here in just 3 years suggest that moderate N deposition in N-limited arid areas (Noy-Meir, 1973; Zhou et al., 2011, 2014) might actually facilitate and accelerate growth of moss crusts.

The increase in population density of Syntrichia following high N enhancement partially compensated for the decrease in individual plant size and biomass. Interestingly, the increase in moss abundance recorded here stands in contrast to several previous studies. For example, in the western USA, summer irrigation and N application interacted to mitigate the effects of decreased shoot numbers of S. caninervis (Stark et al., 2011). Increased N deposition in acidic grassland of the Derbyshire Dales in the UK resulted in a loss of up to 90 % bryophyte cover with no observed recovery (Arróniz-Crespo et al., 2008). A possible explanation for the increased population density observed in this study may be that added N stimulates protonemal growth leading to a trade-off between individual plant biomass and number of individual shoots. This is reminiscent of the analogous strategy of size–density relationships in vascular plants as a result of competition for resources (Chu et al., 2008) and, in mosses, may be a mechanism that facilitates survival under changing environments. However, when biomass accumulation based on specific area is taken into account, population biomass decreased with large amounts of added N. Changes in bryophyte population biomass have also been observed in other studies; increasing atmospheric N deposition was accompanied by decreased stem volumetric density (g dm–3) in Sphagnum (Bragazza et al., 2004), and 2 years of N addition led to increased total bryophyte biomass in a coastal dune grassland in the UK (Plassmann et al., 2009).

High N additions reduced soil pH and increased EC, while no significant effects were found for soil OM, total N and P, and available N. Acidification generated by N deposition has also been reported in other studies. For example, in semi-arid Mediterranean environments of central Spain, increasing N deposition increased soil inorganic N availability and acidified the soil (Ochoa-Hueso et al., 2014).

Sensitivity of moss physiological activity to N addition

Small amounts of added N increased the concentrations of both chlorophyll b and total chlorophyll, but did not affect chlorophyll fluorescence. In contrast, these parameters were suppressed by high N. These results are consistent with our hypothesis that photosynthetic activity in S. caninervis would be stimulated by small amounts of added N, and suppressed by higher amounts.

Osmotic indices may reflect physiological status in response to added N. The decrease in soluble sugar concentrations we recorded suggests that added N may alleviate the effect of N limitations on moss growth. Similar results have been observed for vascular plants. For example, increased levels of N have been correlated with lower soluble sugar and proline concentrations for two annual vascular plants in the Gurbantunggut Desert (Zhou et al., 2011). However, in this study, proline concentrations were not affected by added N. The trend observed in the concentration of soluble protein, increasing at low amounts of added N (up to N1) and decreasing with higher concentrations (N1·5 and N3), probably reflects the fact that while N is directly absorbed by the moss leaf where it is readily transformed into protein (Soares and Pearson, 1997), large amounts of N impair leaf metabolic function, thus disrupting protein synthesis.

The general trend in antioxidant enzyme activities observed in Syntrichia to added N, although we recorded a statistically significant response only in POD and not SOD and CAT activities, parallels that of the growth parameters, i.e. increasing at low concentrations of added N and increasing with higher amounts. POD, SOD and CAT are important antioxidative defence enzymes, protecting cells from the damage induced by free radicals when cells are under oxidative stress, for example as a result of drought, salinity, chilling and metal toxicity (Sharma et al., 2012; Hashempour et al., 2014). Antioxidant enzyme activity usually increases when plants are under stress, e.g. in cyanobacterial crusts, increases in SOD, POD and CAT activity prevent oxidative damage from UV-B radiation (Xie et al., 2009), but may also decrease if the antioxidative enzyme system becomes disrupted, e.g. moss crusts under enhanced UV-B radiation (Hui et al., 2012). N limitation can also cause oxidative stress. N deficiency elicits oxidative damage in tobacco and rice, with a corresponding increase in SOD and CAT activity and in the concentration of malondialdehyde (Huang et al., 2004; Rubio-Wilhelmi et al., 2011). In the present study, N deficiency was alleviated by moderate N addition, and POD, SOD and CAT activity decreased. However, large amounts of added N (N1·5 and N3 treatments), which had a negative effect on moss growth and physiology, resulted in increased activity of these antioxidative enzymes. Therefore, POD, SOD and CAT activity in S. caninervis may be used a useful indicator of N pollution in desert soils.

Critical levels of N deposition on growth of moss crusts and implications for desert ecosystems

Mosses are particularly sensitive to environmental change and thus are suitable bioindicators for environmental assessments (Ochoa-Hueso and Manrique, 2013; Ochoa-Hueso et al., 2014). Critical levels of pollution are defined as the concentrations of pollutants in the atmosphere which have direct adverse effects on receptors such as plants and ecosystems (Krupa, 2003). For example, physiological responses to N of the moss Racomitrium lanuginosum indicate that the critical level on heathlands is exceeded by addition of 1 g N m–2 year–1 (Pearce et al., 2003).

Nitrate reductase, phosphomonoesterase, and tissue N and K+ concentrations in the moss Pleurochaete squarrosa from a semi-arid area of Central Spain were saturated at only 1 g N m–2 year–1 above the background levels, whereas chlorophyll a and lutein increased even at amounts of up to 6 g N m–2 year–1 (Ochoa-Hueso and Manrique, 2013). The addition of only 1 g N m–2 year–1 (plus severe desiccation stress) promoted chlorosis and inhibited regeneration responses of mosses from arid areas of western USA (Stark et al., 2011). Overall, most moss species appear to respond negatively to N addition at rates of up to 1 g N m–2 year–1. From the present study, we conclude that the relatively safe rate for N deposition on moss BSCs in the Gurbantunggut Desert is up to 0·5 g N m–2 year–1; above this rate, N deposition will almost certainly have a negative effect. It must be underlined that this rate is lower than that for cyanobacterial and lichen BSCs (Zhou et al., 2015) and the range of critical levels reviewed by Krupa (2003), who concluded that 0·5–1 g N m–2 year–1 total N deposition would not affect cryptogam ecosystems.

We conclude that if N pollution continues to increase, this may lead to the decline and the disappearance of moss BSCs from the Gurbantunggut Desert.

ACKNOWLEDGEMENTS

This study was jointly supported by the National Basic Research Program (2014CB954202), the National Natural Science Foundation of China (41471251, 41571256), the West Light Foundation of the Chinese Academy of Sciences (XBBS-2014-20) and the Youth Innovation Promotion Association CAS (2015356). We thank Lin Wu, Ye Tao and Guodong Li for their hard work in the field. We are grateful to two anonymous reviewers and the handling editor Dr Silvia Pressel, who greatly contributed to improving the article.

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