Ammonium first: natural mosses prefer atmospheric ammonium but vary utilization of dissolved organic nitrogen depending on habitat and nitrogen deposition Xue-Yan Liu 1,2 , Keisuke Koba 2 , Akiko Makabe 2 , Xiao-Dong Li 1 , Muneoki Yoh 2 and Cong-Qiang Liu 1 1 State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550002, China; 2 Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu 1838509, Japan Author for correspondence: Keisuke Koba Tel: +81 42 367 5951 Email: [email protected]Received: 24 January 2013 Accepted: 18 March 2013 New Phytologist (2013) 199: 407–419 doi: 10.1111/nph.12284 Key words: ammonium, atmospheric nitrogen deposition, denitrifier, dissolved organic nitrogen, moss, nitrogen preference, soil nitrogen availability, stable nitrogen isotope. Summary Mosses, among all types of terrestrial vegetation, are excellent scavengers of anthropogenic nitrogen (N), but their utilization of dissolved organic N (DON) and their reliance on atmospheric N remain uncharacterized in natural environments, which obscures their roles in N cycles. Natural 15 N abundance of N sources (nitrate (NO 3 ), ammonium (NH þ 4 ) and DON in deposi- tion and soil) for epilithic and terricolous mosses was analyzed at sites with different N deposi- tions at Guiyang, China. Moss NO 3 assimilation was inhibited substantially by the high supply of NH þ 4 and DON. Therefore, contributions of NH þ 4 and DON to moss N were partitioned using isotopic mass-balance methods. The N contributions averaged 56% and 46% from atmospheric NH þ 4 , and 44% and 17% from atmospheric DON in epilithic and terricolous mosses, respectively. In terricolous mosses, soil NH þ 4 and soil DON accounted for 16% and 21% of bulk N, which are higher than current estimations obtained using 15 N-labeling methods. Moreover, anthropogenic NH þ 4 deposition suppressed utilization of DON and soil N because of the preference of moss for NH þ 4 under elevated NH þ 4 deposition. These results underscore the dominance of, and preference for, atmospheric NH þ 4 in moss N utilization, and highlight the importance of considering DON and soil N sources when esti- mating moss N sequestration and the impacts of N deposition on mosses. Introduction Nitrogen (N) is an important plant nutrient. Since the 19th Century, anthropogenic N deposition has been increasing globally, triggering major changes in terrestrial ecosystems, including aspects of N and carbon (C) dynamics (Aber et al., 1998; McLauchlan et al., 2007; Lovett & Goodale, 2011) and floristic diversity (Bobbink et al., 2010; De Vries et al., 2010). Therefore, it is important to gain insights into how the N inputs change the N dynamics in terrestrial ecosystems (Phoenix et al., 2012; Templer et al., 2012). Ascertaining changes of the ecosystem N dynamics with increased N inputs is also necessary to estimate changes in ecosystem functions (Manning et al., 2006; Pardo et al., 2012). The preferences of plants and microbes for different N forms among ammonium (NH þ 4 ), dissolved organic N (DON), and nitrate (NO 3 ) play an important role in determining the ecosys- tem N dynamics (Northup et al., 1995; Lovett & Mitchell, 2004) and in determining the fates of N input into natural ecosystems (Durka et al., 1994; Liu et al., 2012c). Nevertheless, it remains extremely difficult to evaluate plant N preferences, partly because these three forms of soil N have different physical, chemical, and biological characteristics, and therefore have different availabilities to plants (Kaye & Hart, 1997; Abaas et al., 2012). Nitrogen utilization by plants includes N uptake and assimila- tion. For N assimilation, the incorporation of different N forms into plant biomass differs in assimilation costs (Gutschick, 1981; Clarkson, 1985; Li et al., 2013). The assimilation cost of amino acids, a small but important DON component, is expected to be lower than that of NH þ 4 , which must be attached to a C skeleton before use, and much lower than that of NO 3 , which requires additional reduction steps to NH þ 4 (Clarkson, 1985; Bloom et al., 1992; Chapin et al., 1993). These differences partially explain the preferences for NH þ 4 or amino acids observed in vas- cular plants when different N forms are supplied in equal doses (Kronzucker et al., 1997; Houlton et al., 2007; Wang & Macko, 2011). However, the roots of vascular plants actually take up N through different pathways (e.g. mycorrhizal symbionts) and from different soil depths with different availabilities (McKane et al., 2002; Kohzu et al., 2003), which obscures the real reasons for N preferences in natural vascular plants. In contrast to vascu- lar plants, mosses lack a rooting system. With leaves only one cell thick and no cuticular barrier in most taxa, moss cells are exposed Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust New Phytologist (2013) 199: 407–419 407 www.newphytologist.com Research
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Ammonium first: natural mosses prefer atmospheric ammoniumbut vary utilization of dissolved organic nitrogen depending onhabitat and nitrogen deposition
1State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550002, China; 2 Institute of Agriculture, Tokyo University of
Agriculture and Technology, Fuchu 1838509, Japan
Author for correspondence:Keisuke KobaTel: +81 42 367 5951
� Mosses, among all types of terrestrial vegetation, are excellent scavengers of anthropogenic
nitrogen (N), but their utilization of dissolved organic N (DON) and their reliance on atmospheric
N remain uncharacterized in natural environments, which obscures their roles in N cycles.� Natural 15N abundance of N sources (nitrate (NO�
3 ), ammonium (NHþ4 ) and DON in deposi-
tion and soil) for epilithic and terricolous mosses was analyzed at sites with different N deposi-
tions at Guiyang, China. Moss NO�3 assimilation was inhibited substantially by the high supply
of NHþ4 and DON. Therefore, contributions of NHþ
4 and DON to moss N were partitioned
using isotopic mass-balance methods.� The N contributions averaged 56% and 46% from atmospheric NHþ
4 , and 44% and 17%
from atmospheric DON in epilithic and terricolous mosses, respectively. In terricolous mosses,
soil NHþ4 and soil DON accounted for 16% and 21% of bulk N, which are higher than current
estimations obtained using 15N-labeling methods. Moreover, anthropogenic NHþ4 deposition
suppressed utilization of DON and soil N because of the preference of moss for NHþ4 under
elevated NHþ4 deposition.
� These results underscore the dominance of, and preference for, atmospheric NHþ4 in moss
N utilization, and highlight the importance of considering DON and soil N sources when esti-
mating moss N sequestration and the impacts of N deposition on mosses.
Introduction
Nitrogen (N) is an important plant nutrient. Since the 19thCentury, anthropogenic N deposition has been increasing globally,triggering major changes in terrestrial ecosystems, including aspectsof N and carbon (C) dynamics (Aber et al., 1998; McLauchlanet al., 2007; Lovett & Goodale, 2011) and floristic diversity(Bobbink et al., 2010; De Vries et al., 2010). Therefore, it isimportant to gain insights into how the N inputs change the Ndynamics in terrestrial ecosystems (Phoenix et al., 2012; Templeret al., 2012). Ascertaining changes of the ecosystem N dynamicswith increased N inputs is also necessary to estimate changes inecosystem functions (Manning et al., 2006; Pardo et al., 2012).The preferences of plants and microbes for different N formsamong ammonium (NHþ
4 ), dissolved organic N (DON), andnitrate (NO�
3 ) play an important role in determining the ecosys-tem N dynamics (Northup et al., 1995; Lovett & Mitchell, 2004)and in determining the fates of N input into natural ecosystems(Durka et al., 1994; Liu et al., 2012c). Nevertheless, it remainsextremely difficult to evaluate plant N preferences, partly becausethese three forms of soil N have different physical, chemical, and
biological characteristics, and therefore have different availabilitiesto plants (Kaye & Hart, 1997; Abaas et al., 2012).
Nitrogen utilization by plants includes N uptake and assimila-tion. For N assimilation, the incorporation of different N formsinto plant biomass differs in assimilation costs (Gutschick, 1981;Clarkson, 1985; Li et al., 2013). The assimilation cost of aminoacids, a small but important DON component, is expected to belower than that of NHþ
4 , which must be attached to a C skeletonbefore use, and much lower than that of NO�
3 , which requiresadditional reduction steps to NHþ
4 (Clarkson, 1985; Bloomet al., 1992; Chapin et al., 1993). These differences partiallyexplain the preferences for NHþ
4 or amino acids observed in vas-cular plants when different N forms are supplied in equal doses(Kronzucker et al., 1997; Houlton et al., 2007; Wang & Macko,2011). However, the roots of vascular plants actually take up Nthrough different pathways (e.g. mycorrhizal symbionts) andfrom different soil depths with different availabilities (McKaneet al., 2002; Kohzu et al., 2003), which obscures the real reasonsfor N preferences in natural vascular plants. In contrast to vascu-lar plants, mosses lack a rooting system. With leaves only one cellthick and no cuticular barrier in most taxa, moss cells are exposed
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directly to environmental N sources (Glime, 2007). Moss Nuptake might not cause substantial preference because nutrientscan enter moss tissues easily through cation exchange and theproton (H+) pump (for NHþ
4 and amino acids) and throughcotransport (for NO�
3 ) with positively charged ions (Raven et al.,1998; Glime, 2007). When the supply rates of different N formswere identical, N preferences observed in mosses were derivedmainly from the assimilation (Pearce et al., 2003; Wiedermannet al., 2009). Nevertheless, no suitable method exists to confirmthe N preference of mosses in natural environments with variableavailabilities of different N forms. It is therefore unknown if mossN preferences will vary along with the N source availability.
The natural abundance of N isotope (d15N; the 15N : 14N ratioexpressed relative to atmospheric N2) in natural plants can inte-grate available N sources and physiological processes (H€ogberg,1997; Robinson, 2001; Craine et al., 2009). Compared with theisotopic labeling method (Wanek & Zotz, 2011), d15N analysisavoids artificial N addition and therefore presents no risk ofchanging the soil N pool and plant N-uptake kinetics. In particu-lar, using the d15N of mosses to evaluate their N sources is likelyto yield better results than for vascular plants, because mostmosses have no cuticular barrier, stomatal regulation, or rootmycorrhizal mediation, with these organs and regulations appar-ent 15N fractionations can be expressed during N acquisition andreallocation in vascular plants (Handley & Scrimgeour, 1997;Evans, 2001; Gebauer & Meyer, 2003; Hobbie & H€ogberg,2012). Consequently, the d15N analysis of mosses can revealdominant N sources and or N preferences in N assimilation. Instudies in China, for example, the d15N of mosses on bare rockshowed low values in regions where inorganic N deposition wasdominated by NHþ
4 (Liu et al., 2008; Xiao & Liu, 2011). How-ever, the extent to which mosses rely on NHþ
4 and the extent towhich they use NO�
3 and DON remain open questions. Suchknowledge is of global importance because NHþ
4 is dominant inN deposition in most regions of the world (Pearson & Stewart,1993; Stevens et al., 2011). The preferred utilization of NHþ
4
over NO�3 has also been emphasized for vascular plants because
NHþ4 preference interacts closely with ecosystem processes and
functioning (Houlton et al., 2007; Kahmen et al., 2008;Boudsocq et al., 2012). Measurements of stable isotopes (d15Nand d18O) of tissue NO�
3 in mosses (Liu et al., 2012a) revealedthat moss NO�
3 assimilation is inducible when NO�3 is the sole
N source (Liu et al., 2012b), but moss NO�3 assimilation was
found to be negligible when the supply rate of reduced dissolvedN (RDN; NHþ
4 plus DON) was significantly higher than that ofNO�
3 in natural environments (e.g. Liu et al., 2012c). This lowassimilation of NO�
3 in mosses across different habitats resultedfrom the inhibition of nitrate reductase activity (NRA) by thehigh supply rate of RDN (detailed in Liu et al., 2012c; detailedmechanisms are reviewed by Dortch, 1990). Consequently, mea-suring d15N of NHþ
4 and DON in deposition allows further par-titioning of moss NHþ
4 and DON assimilation, an exploration ofNHþ
4 preference in epilithic mosses, and an evaluation of the bio-availability of DON in RDN-dominated deposition.
The exploration of DON assimilation in mosses is importantfor two main reasons. First, present knowledge of moss N
sequestration and N deposition effects on mosses largely relatesto inorganic N (Paulissen et al., 2004; Bragazza et al., 2005;Gundale et al., 2011) and N2 fixation (for feather mosses;DeLuca et al., 2008; Ackermann et al., 2012). However, theassimilation of DON and its interaction with inorganic N (NHþ
4
and NO�3 ) assimilation remain unclear. This poses the question
of whether the importance of inorganic N deposition to terres-trial mosses has been overrated because of neglect or underesti-mation of the true contribution of DON. Second, exploration ofDON assimilation can expand the characterization of moss Npreference (Soares & Pearson, 1997; Arr�oniz-Crespo et al.,2008). Mosses prefer NHþ
4 when using inorganic N sourcesbecause of the high energy cost of NO�
3 reduction and thepotential for avoiding excessive NHþ
4 accumulation (Pearson &Stewart, 1993; for vascular plants, Kronzucker et al., 2001).However, it is unclear whether there is a moss N preference forNHþ
4 over DON because in natural environments thebioavailability of DON has not been fully characterized.Through 15N labeling, some laboratory and field studies haverevealed considerable utilizations of amino acids in mosses andinfluences of amino acid accumulation on inorganic N metabo-lism (Baxter et al., 1992; Nordin & Gunnarsson, 2000). Forsumet al. (2006) applied 50 kg N ha�1 yr�1 (NHþ
4 : NO�3 : gly-
cine = 1 : 1 : 1) to mosses and found a clear preference for aminoacid N over NO�
3 , although the assimilation of glycine remainedlower than that of NHþ
4 . Similarly, Wanek & P€ortl (2008)reported that the uptake rates of NHþ
4 or glycine were two timeshigher than those of NO�
3 . By15N-labeling of NO�
3 , NHþ4 , ala-
nine, and glutamic acids, Wiedermann et al. (2009) found thatmosses preferred NHþ
4 and DON, with very low uptake of NO�3
under different levels of N deposition. These studies demon-strated that, in natural conditions, NO�
3 is a negligible N sourcefor mosses, but amino acids may contribute substantially to mossN sequestration. It is noteworthy that amino acids account foronly a small proportion of DON, and that the bioavailable frac-tion of DON is expected to be larger than that of amino acids(Neff et al., 2002). To date, only the d15N of ‘bulk’ DON can bemeasured routinely (Knapp et al., 2005; Koba et al., 2010a,b;Lachouani et al., 2010), but this allows exploration of whetherplants indeed prefer NHþ
4 when isotopes of major dissolved N innatural environments are characterized (Houlton et al., 2007;Kahmen et al., 2008; Takebayashi et al., 2010). Thereby, theimportance of DON in moss N assimilation can be estimated.
Four sets of moss–soil systems (mosses on bare rock, mossesgrowing on the soil of the rock surface, and terricolous mossesin open fields and on forest floors) under high (urban area;21 kg N ha�1 yr�1) and low (suburban and rural areas;10–12 kg N ha�1 yr�1) N deposition rates were investigated inthe Guiyang area of China. Because of the dominance of NHþ
4
or RDN in total dissolved N (TDN) deposition (Table S1),depleted d15N values have been observed in mosses, especiallyspecies on bare rock (Liu et al., 2008). Moss NRA was inhibitedsubstantially by high atmospherically derived (atm-) RDN.Therefore, a negligible contribution of NO�
3 was found in mossN assimilation in this area (Liu et al., 2012c). By measurementof the pool sizes and d15N signatures of dissolved N (NHþ
4 ,
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DON, and NO�3 ) in soil attached with mosses, the present
work aimed (1) to verify the importance of atm-NHþ4 for
mosses in natural habitats through determination and consider-ation of the contributions of atm-DON and soil N, and (2) toexplore whether mosses prefer atm-NHþ
4 over DON or soil Nby comparing their contributions in mosses with their availabili-ties across the gradient of anthropogenic NHþ
4 (Liu et al.,2008a). Our main hypothesis is that moss N assimilation isdominated by atm-NHþ
4 , not only because of high depositionof atm-NHþ
4 , but also because of the preference for atm-NHþ4
over DON and soil N.
Materials and Methods
Study area and sample collection
The Guiyang area, located in southwestern China, has a typicalsubtropical monsoon climate and an average altitude of 1250 m(a detailed description is given in Supporting Information NotesS1). The wet deposition was collected from December 2008 toSeptember 2009 at an urban site in Guiyang (detailed in NotesS1). In July 2010, epilithic mosses on bare rock (EB), mossesgrowing on the soil of the rock surface (ES), and terricolousmosses in open fields (TO) and on the floor of pine forests (TF),as well as soil attached with mosses (< 3 cm), were collectedalong a northeastern urban (U)–suburban (S)–rural (R) transectin the Guiyang area (sampling sites are shown in Fig. S1). Thesehabitats represent typical habitats of natural mosses. For mosssampling, mature and green tissues at five to 10 subsites werecollected and then mixed as composite samples for each site. Toavoid possible interspecific differences, moss species withuniform or similar morphological traits were considered. Eachsample of mosses on rock (EB and ES) was a mixture ofEurohypnum (mainly Eurohypnum julaceum and Eurohypnumleptothallum (c. mull.) ando), Hypnum (Hypnum plumaeformeWils.), and Haplocladium (mainly Haplocladium microphyllumHedw.), whereas terricolous moss samples included species ofHypnum (mainly Hypnum plumaeforme Wils.), Thuidium(mainly Thuidium cymbifolium (Dozy et Molk.) Dozy et Molk.),and Haplocladium microphyllum Hedw. These species, all pleuro-carpous, have a widespread distribution all over the world. Forsoil sampling, only soil to a depth of 3 cm was collected becauseour observations and experience in the field suggested thatsoil-anchored rhizoids or moss layers were generally 3 cm deep.Mosses on forest floors were sampled from three unmanagedpine (Pinus massoniana Lamb. var) forests. Three sampling plotswere set in each forest. Then mosses and soil (< 3 cm) at three tofive subsites were combined to form one sample in each plot. ForES and TO, eligible soil and moss samples were found to beinsufficient at each sampling site (marked in Fig. S1) for allexperimental analyses. Therefore, samples were mixed forU1 and U2, U3 and U4, and U5 and U6 in urban areas, for S1and S2, S3 and S4, and S5 and S6 in suburban areas, and forR1 and R2 and R3 and R4 in rural areas (Fig. S1). Consequently,the number of replicates was three for samples of ES, TO, andTF.
Experimental analyses
Within 8 h after sampling, sample pretreatments were conductedin the laboratory at the Institute of Geochemistry (CAS), Guiyang,China. Some of the fresh soil was used to determine water contents.The remainder was passed through a 2-mm mesh sieve to removeimpurities and coarse fragments. Some of the sieved soil was usedto determine pH (H2O). The remainder was used immediatelyfor extraction of dissolved inorganic N (DIN: NHþ
4 plus NO�3 )
and DON, with a ratio of 10 g of fresh soil to 100ml of 2M KClsolution. The mixture of soil and KCl solution was shaken for1.5 h, then centrifuged. The supernatant was filtered using a glassfilter (GF/F; Whatman, Maidstone, UK). The KCl salts and filterswere heated to 450°C for 4 h to reduce the N blank before use.A subsample of the sieved soil was dried at 60°C to measure water,C, and N contents as well as d15N values. The preparation of mosssamples has been described by Liu et al. (2012c).
The ball-milled soil samples, diffusion samples of soil NHþ4
(see details in Notes S1), soil extraction solutions (frozen at�20°C) and the sieved fresh soil (at 4°C) were shipped to TokyoUniversity of Agriculture and Technology within 3 d after sam-pling. The methods described by Takebayashi et al. (2010) wereused to incubate soil for determination of net N nitrification andN mineralization rates. The concentrations and stable isotopes ofbulk N in soil, NHþ
4 , NO�3 and TDN in soil extracts were ana-
lyzed using the methods described by Koba et al. (2010a) and byTakebayashi et al. (2010) (details in Notes S1). The RDN wascalculated as the difference between TDN and NO�
3 . DON wascalculated as the difference between TDN and DIN. The d15Nvalues of DON and RDN were calculated, respectively, using thefollowing mass and isotopic balance equations:
d15NDON ¼ fd15NTDN � ½TDN� � d15NNO3� � ½NO�3 �
� d15NNH4þ � ½NHþ4 �g=½DON�:
d15NRDN ¼ fd15NTDN � ½TDN� � d15NNO3�� ½NO�
3 �g=½RDN�:
The d15N was expressed as (Coplen, 2011):
d15N ¼ ðRsample=RstandardÞ � 1;
where R = 15N/14N in samples and the standard (atmosphericN2, and Rstandard = 0.0036765).
Results
Dissolved N forms in soil attached with mosses
Table 1 presents characteristics (pH, C : N ratio, net Nnitrification and mineralization rates) of moss soil. Details areshown in Notes S2. Dissolved N concentrations in the soil of epi-lithic mosses were higher than those in soils of terricolous mosses,with the lowest in soils of forest mosses (Table 1). The soils of
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epilithic mosses showed higher dissolved N concentration inurban than in rural areas (P < 0.05), although such spatial differ-ences were not observed in the soils of terricolous mosses. More-over, the pool of RDN was significantly (P < 0.05) larger thanthat of NO�
3 in moss soil, showing much higher RDN: NO�3
ratios (6–71; Table 1) than that in wet deposition (4.9; Table S1).Variations of d15N in bulk N (0.6–5.2&), in TDN (2.2–
6.4&), and in RDN (2.8–6.9&) showed no clear pattern inurban or rural areas (Table 1). The d15N values of TDN(4.6� 1.2&) and DON (3.8� 2.0&) were generally morepositive than that of bulk N (2.7� 1.4&). Soil NHþ
4 showedthe most positive d15N values (2.5–11.2&), in contrast to thelowest d15N signatures of NO�
3 (�8.9 to 2.1&) (Fig. 1). In gen-eral, soil d15N-NO�
3 was lower at urban sites than at rural sites,and lower in soil in open fields than in soil in pine forests(Fig. 1).
Moss bulk N and d15N signatures
Moss N concentration varied from 14.1 to 26.5 mg g�1, differingamong habitats (mean values from Liu et al., 2012c are listed inTable S2) and generally increased under elevated N deposition inurban areas. Epilithic mosses with soil had higher N than mosseson bare rock, although mosses in pine forests had the lowest N.Moss d15N ranged between –12.9& and –1.3& (Fig. 1 andTable S2; Liu et al., 2012c). Overall, the d15N was lower in urbanmosses than in rural mosses, and lower in mosses on bare rockthan in mosses growing on soil.
Calculations of the deposited N and soil N contributions tomoss bulk N
The assimilation of NO�3 was negligible as a result of the
inhibition of moss NRA by RDN associated with much higherdeposition of RDN than that of NO�
3 (described in Liu et al.,2012c). Therefore, mosses in our study area mainly assimilatedNHþ
4 and DON. Consequently, the proportional contributions(f, expressed as percentages hereafter) of atm-DON (fatm-DON)and atm-NHþ
4 (fatm-NH4+ = 1 � fatm-DON) to bulk N of mosseson bare rock (Nmoss-rock) were calculated using a two-sourcemixing model:
soil-NHþ4 and soil-DON). The total contribution of soil-RDN
and deposited RDN can be estimated using a two-source mixingmodel (detailed in Notes S3; shown in Fig. S2). The proportionalcontributions of explicit N species are calculable using the ‘Iso-Source’ model (Phillips & Gregg, 2003) (Eqn. 3).
The entry of N into moss tissues has no substantial 15N frac-tionation (Liu et al., 2012b). Therefore, this model iterativelygenerates source isotopic mixtures of which the proportions(f ) sum to 1 (fatm-NH4+ + fatm-DON + fsoil-NH4+ + fsoil-DON = 1). Itcompares each calculation against a known mixture (d15Nmoss-
soil) and retains only those mixtures that satisfy the known valuewithin some mass balance tolerance, as defined using a data set offeasible solutions. Although this model can only generate feasiblesolutions, it nevertheless provides a systematic means of con-straining the attribution of N sources in an underdetermined
Fig. 1 The natural abundance of nitrogen (N)isotope (d15N) signatures of moss N,dissolved N (NHþ
4 , dissolved organic N(DON) and NO�
3 ) in wet deposition andmoss soil in the Guiyang area. EB, epilithicmosses on bare rock; ES, mosses growing onthe soil of the rock surface; TO, terricolousmosses in open fields; TF, terricolous mossesin pine forests. Solid and dashed lines withinthe boxes show the median and the mean,respectively. The box boundary shows the25th and 75th percentiles. Dots aside fromthe caps indicate each outlier of data points.The d15N of NHþ
4 in deposition is after Xiaoet al. (2012). The d15N of mosses, totaldissolved N (TDN) and NO�
3 in depositionfollow Liu et al. (2012c). The d15N of DON indeposition was calculated using the isotopicmass -balance equation (d15NDON ={d15NTDN9 [TDN] – d15NNO3-9 [NO�
3 ] –d15NNH4+9 [NHþ
4 ]}/[DON]) based onmonthly mean concentrations and d15Nvalues. Details of sampling and analyses ofdeposited N are provided in Liu et al.
(2012c).
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system. In our case, the calculated mixtures reflecting combina-tions of the d15N values of atm-NHþ
4 , atm-DON, soil-NHþ4 ,
soil-DON, and moss N, we applied a mass balance tolerance of0.2%. The original data output from the model for all replicatesamples is presented in Fig. S3.
For mosses on bare rock, the fatm-NH4+ (56� 13%; 44–90%)was higher than fatm-DON (44� 13%; 10–55%) (Table 2) andsignificant differences (P < 0.05) were found between urban andrural areas (Fig. 2a). In urban areas, the fatm-NH4+ was 68� 15%,which decreased to 49� 1% and 56� 3% in suburban and rural
areas, respectively, with a corresponding increase in the fatm-DON
(Fig. 2a).On average, mosses on soil derived more N from deposition
(63� 8%; fatm-N = fatm-NH4+ + fatm-DON) than from soil(37� 8%; fsoil-N = fsoil-NH4+ + fsoil-DON), and more N fromNHþ
Reported data are means� SD. fatm-N = fatm-NH4+ + fatm-DON; fsoil-N = fsoil-NH4+ + fsoil-DON; ftotal-NH4+ = fatm-NH4+ + fsoil-NH4+; ftotal-DON = fatm-DON + fsoil-DON.Mosses on soil include ES, TO and TF. NO�
3 , nitrate; NHþ4 , ammonium; DON, dissolved organic N; atm-N, N from atmospheric deposition; soil-N, N from
soil; ES, mosses growing on the soil of the rock surface; TO, terricolous mosses in open fields; TF, terricolous mosses in pine forests.
(a) (b)
(c) (d)
Fig. 2 Comparisons of the proportionalcontributions of atmospherically derived(atm-) NHþ
4 with those of (a) atmosphericallyderived total dissolved nitrogen (atm-DON)in mosses on bare rock (n = 6 for urban andsuburban sites; n = 5 for rural sites), (b) atm-DON in mosses on soil (n = 9), (c) total DON(atm-DON plus soil-DON) in mosses on soil(n = 9), and (d) total soil N (soil NHþ
4 plus soil-DON) in mosses on soil (n = 9). Mosses onsoil include mosses growing on the soil of therock surface (ES), terricolous mosses in openfields (TO), and terricolous mosses on floorsof pine forests (TF). Reported aremeans� SD. Mean values of percentageranges (for each replicate sample) outputfrom the two-source mixing model and the‘IsoSource’ model were used.
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mean contributions were, respectively, 17� 5%, 16� 4%, and21� 7% (Table 2). Similar to the pattern for mosses on barerock (Fig. 2a), the fatm-NH4+ in all mosses on soil showed a cleardecrease from the urban (56� 11%) to suburban (42� 4%) andrural areas (40� 4%) (Fig. 2b). By contrast, the contributions ofatm-DON (fatm-DON; Fig. 2b), total DON (Fig. 2c) and soil N(Fig. 2d) showed an opposite pattern to that of atm-NHþ
4 fromurban to rural areas. Moreover, neither proportional nor real con-tributions of soil N (soil-NHþ
4 and soil-DON) in Nmoss (realcontribution = proportional contribution9Nmoss; in mg N g�1
DW) responded clearly to soil N availability (Fig. S4).
Estimation of moss preference for NH+4 versus DON
Plant N preference for a given N source ‘A’ over the other Nsource ‘B’ (bA) can result from both uptake and assimilation pro-cesses (Boudsocq et al., 2012; Li et al., 2013). The N preferenceduring uptake is related to external abundance and mobility ofspecific N species, and root properties, whereas the N preferenceduring assimilation is associated with the energy cost of specificN incorporation (Gutschick, 1981; Clarkson, 1985; Bloom et al.,1992). Previous to this work, the uptake preference was oftenevaluated using the ratio of N uptake rates between ‘A’ and ‘B’when they were equally available (e.g. preference for NHþ
4 overNO�
3 ; Chapin et al., 1986; Kronzucker et al., 1997; N€asholmet al., 1998; preference for glycine over NHþ
4 ; Chapin et al.,1993; Kielland, 1997; Raab et al., 1999). Moss N uptake mightnot result in preferences because mosses have very simple tissuestructure. Moreover, cation exchanges (for NHþ
4 and aminoacids) occur simultaneously with anion cotransport (for NO�
3 ).Consequently, to ascertain the differences between the assimila-tion and relative availability of specific N forms, we can exploreN preference although in natural conditions N forms are sup-plied with variable ratios. A preferential assimilation will result ina higher contribution of ‘A’ in overall assimilation of ‘A’ and ‘B’than the proportional contribution of ‘A’ in the total availabilityof ‘A’ and ‘B’. Accordingly, the preference for atm-NHþ
4 overatm-DON (batm-NH4+) can be inferred for all mosses as the dif-ference between (the proportional contribution of atm-NHþ
4
assimilation in atm-RDN assimilation) and (the proportionalcontribution of atm-NHþ
4 in atm-RDN; 62%; Table S1).
batm�NH4þ ¼ fatm�NH4þ=fatm�RDN � 0:62 Eqn 4
The preference for soil-NHþ4 over soil-DON (bsoil-NH4+) can
be described for mosses on soil as the difference between the pro-portional contribution of soil-NHþ
4 assimilation in soil-RDNassimilation and the proportional contribution of soil-NHþ
where fatm-RDN = fatm-NH4+ + fatm-DON and fsoil-RDN = fsoil-NH4+ + fsoil-DON. The fatm-NH4+, fatm-DON, fsoil-NH4+ and fsoil-DON
were calculated using Eqns 1–3. The averaged values of each rep-licate sample were used for the bNH4+ calculation. Positive values,0, and negative values of b, respectively, show a preference forNHþ
4 , no preference, and a preference for DON.For atmospheric N sources, mosses in all habitats had the high-
est batm-NH4+ values in the urban area (Fig. 3a). In suburban andrural areas, the batm-NH4+ value generally decreased; in particular,the batm-NH4+ values became negative for mosses on bare rock(Fig. 3a). By contrast, for soil N sources, the bsoil-NH4+ valueswere negative for terricolous mosses in open fields in the urbanarea, whereas mosses in pine forests had positive bsoil-NH4+ values(Fig. 3b). A negative correlation was found between the batm-
NH4+ and bsoil-NH4+ for mosses on soil (Fig. 4).
Discussion
Contributions of atm-NH+4 and atm-DON in mosses on
bare rock
With no N supply from substrates, %N and d15N in mosses onbare rock are good indicators of anthropogenic N deposition(Pearson et al., 2000). Low moss d15N observed in the Guiyangarea reflected the dominance of RDN (low d15N; Table S1) in Ndeposition. Lower moss d15N in the urban than in the rural area(Fig. 1) was attributed mainly to high NHþ
4 from urban sewage/waste NH3 emission, which created an urban–rural gradient ofanthropogenic N deposition and which was more 15N-depletedthan NHþ
4 from soil/fertilizer NH3 in rural areas (Liu et al.,2012c).
Calculations based on the two-source mixing model revealedhigher contributions from atm-NHþ
4 (56%) than from atm-DON (44%) to N in mosses on bare rock, particularly in urbanareas (fatm-NH4+ = 68%) (Table 2; Fig. 2a). However, in suburbanand rural areas where anthropogenic NHþ
4 deposition was low,the contribution from atm-DON became higher than in theurban area (Fig. 2a). Attention therefore must be devoted to thecontribution of atmospheric DON to the moss N economy.Otherwise, the ecophysiological impacts of anthropogenic atm-NHþ
4 deposition on mosses can be overestimated substantially inour study area. Moreover, mosses can adjust the N-assimilatingregime in response to anthropogenic N deposition (Wiedermannet al., 2009). The evaluation of NHþ
4 preference using batm-NH4+
revealed that urban mosses on bare rock preferred atm-NHþ4 over
atm-DON (0 < batm-NH4+ < 1; Fig. 3a). Because both NHþ4 and
DON can be absorbed into moss cells through cation exchangeand the proton (H+) pump, the uptake might not result in asubstantial preference. The observed atm-NHþ
4 preference wasgenerated mainly from assimilation associated with inherent Ndemand, and potentially with the purpose of reducing impacts ofexcessive NHþ
4 accumulation on moss growth (Limpens &Berendse, 2003), as evidenced by higher efficiencies of intra-plantNHþ
4 assimilation than assimilation of other N forms (Pearson& Stewart, 1993; Kronzucker et al., 2001). This mechanism wassupported in particular by higher batm-NH4+ for mosses underhigh NHþ
4 pollution in the urban area (Fig. 3a). In rural areas,the low anthropogenic NHþ
4 (Liu et al., 2012c) and the supply of
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New Phytologist� 2013 New Phytologist TrustNew Phytologist (2013) 199: 407–419
atm-NHþ4 might not satisfy moss N demand. For these reasons,
epilithic mosses showed greater reliance on atm-DON andshowed little or no preference for atm-NHþ
4 (Fig. 3a). Accord-ingly, the N assimilation in epilithic mosses was dominated byatm-NHþ
4 because the preference for NHþ4 was associated with a
higher rate of NHþ4 deposition. The atm-DON contributed a
substantial fraction of moss N assimilation, although lowerfatm-DON in the rural area than fatm-NHþ
4in the urban area showed a
compromise of atm-DON assimilation to atm-NHþ4 assimilation
(Fig. 3a). Similarly a down regulation of elevated anthropogenicDIN deposition on moss N2 fixation has been observed in feathermosses of boreal regions (DeLuca et al., 2008; Ackermann et al.,2012).
Partitioning of atm-N and soil-N contributions in mossesgrowing on soil
The utilization of soil N sources, which were generally more15N-enriched than those from atmospheric deposition, caused
higher d15N in mosses on N-available substrates than in those onbare rock (Fig. 1). The low net N rates of acidic soil in pine for-ests resulted in low soil N availability to forest mosses. Conse-quently, bulk N and tissue NO�
3 (Liu et al., 2012c) were lower inforest mosses than in other mosses (Tables 1, S4). This raised thequestion of whether atmospheric N is still important for mossesthat have opportunities to use soil N sources, especially underhigh soil N availability. Moreover, lower C : N, higher soil NO�
3
and nitrification rates, and lower d15N of soil NO�3 were
observed in urban than in rural areas (Fig. 1, Table 1). Theseresults demonstrated that N availability and cycling processes inunderlying soils showed a response to elevated N deposition,although direct atmospheric N inputs were largely retained bymoss layers. It is therefore necessary to ascertain whether the utili-zation of soil N in mosses will increase when N supply from bothsoil and deposition is elevated by anthropogenic N pollution.
Showing consistency with mosses on bare rock, the NO�3
assimilation in mosses on soil was inhibited because RDN wasmuch higher than NO�
3 in both deposition and soil (Tables 1,
(a)
(b)
Fig. 3 Preference (b) for (a) atmospherically derived (atm-) NHþ4 over atmospherically derived total dissolved nitrogen (atm-DON) (batm-NH4+; Eqn 4), and
(b) soil-NH4+ over soil-DON (bsoil-NH4+; Eqn 5) in mosses of different habitats. Positive b values denote a NHþ
4 preference. Negative values show a DONpreference; b = 0 shows no preference (dashed lines). Solid and dotted lines within the boxes denote the median and the mean, respectively.
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Research
NewPhytologist414
S1; detailed in Liu et al., 2012c). Provided that mosses had thecapability to assimilate NO�
3 , the fNO3– (mean > 38%) wouldshow higher percentages than the fNH4+ (data not shown; calcu-lated using IsoSource). Such a greater reliance on NO�
3 than onNHþ
4 and preference for NO�3 over NHþ
4 is unlikely in our studyarea. First, the inhibitory effect of NHþ
4 on NO�3 utilization is
well known to prevail among microbes (Rice & Tiedje, 1989),phytoplankton (reviewed by Dortch, 1990), and vascular plants(Kronzucker et al., 1999; Aslam et al., 2001; Wang & Macko,2011). Evidence from phytoplankton showed that the inhibitoryeffect occurs at NHþ
4 concentrations higher than c. 1 lM (a con-centration much lower than NHþ
4 concentrations in precipitationand soil solutions) (Dortch, 1990). Second, many 15N-tracerexperiments have reported extremely low NO�
3 utilization inmosses, even at the same concentrations as other N sources(Soares & Pearson, 1997; Forsum et al., 2006; Wanek & P€ortl,2008; Wiedermann et al., 2009). In our study area, we inferredthat mosses trapped NO�
3 and that the storage of NO�3 depended
on external availability, but NO�3 assimilation did not occur to a
substantial degree under higher deposition of RDN (Liu et al.,2012b,c). Therefore, the partitioning of soil and deposited Nsources was conducted on NHþ
4 and DON for mosses on soil(Figs 2, S2).
There are two main implications of our results. First, terrico-lous mosses did rely more on N from deposition (63%) thanfrom soil (Table 2), but the mean soil N contribution of 37%(Table 2) was much higher than the current estimation based onshort-term (7 d) and solely inorganic 15N additions(NHþ
4 : NO�3 = 1 : 1) in mat-forming mosses (2–9%; Ayres et al.,
2006). Evaluation of moss N sequestration and N depositioneffects should therefore consider the assimilation of soil-derivedN. Neglecting this source might result in considerable overstate-ment of the importance and effects of N deposition. Moreover,the contribution of soil N did not change with soil N availability
(Fig. S4). Forest mosses, which showed little difference in N%and d15N from tericolous mosses in open fields (suggesting littlecanopy effect at least on deposition d15N in our study; Table S2),had substantially lower soil N availability, but the correspondingcontributions of soil N in forest mosses were comparable to thoseof mosses in open fields (Fig. S3). By contrast, the proportionalcontribution of soil N was even lower in mosses of urban areas(Fig. 2d), where both soil N availability and N deposition wereelevated by anthropogenic NHþ
4 deposition (Liu et al., 2008,2012c). The pattern is also apparent in calculations based ond15N of RDN (Fig. S2 and Notes S3), confirming that atmo-spheric N is still more important for mosses than soil N sources,even when soil N availability is elevated. The contribution of soilN to moss N was not responsive to soil N availability (Fig. S4),but decreased with anthropogenic NHþ
4 deposition (Figs 2d, S3).Second, NHþ
4 was the dominant N form among N speciesassimilated by mosses (Table 2). This dominance is attributableto the dominance of NHþ
4 among available N sources (Tables 1and S1) and/or to the preference for NHþ
4 over DON. The great-est contribution was from atm-NHþ
4 (46%; Table 2, Fig. 2). Thisresult added quantitative and field-based evidence that NHþ
4
deposition plays a major role in altering moss N metabolism andspecies composition (Baxter et al., 1992; Aerts & Bobbink,1999). However, an average 38% of moss N was contributedfrom DON (ftotal-DON; Table 2), suggesting that, in natural habi-tats, DON was a notable N contributor to mosses, although theavailability of coexisting DIN was high. Before this study, theutilization of DON (mainly as amino acid N) was estimatedmainly in vascular plants and was based on isotopic labelingmethods. Using 14C-labeling, the uptake of free amino acids bynonmycorrhizal Eriophorum vaginatum L. was estimated asaccounting for > 60% of the N absorbed by this species in arctictundra (Chapin et al., 1993). By injecting 13C-labeled and 15N-labeled glycine into the organic layer in an old boreal forest, itwas estimated that at least 91%, 64%, and 42% of N was takenup, respectively, in intact glycine in dwarf shrubs (Vacciniummyrtillus L.), grass (Deschampsia flexuosa (L.) Trin.), and conifer-ous trees (Pinus sylvestris L. and Picea abies (L.) Karst.) (N€asholmet al., 1998). Additional evidence obtained from arctic moss(Sphagnum rubellum Wils.; Kielland, 1997), subalpine/alpineand temperate species (Raab et al., 1999), and also agriculturalplants (N€asholm et al., 2000) revealed a widespread capability toutilize DON among plants, irrespective of the type of mycorrhi-zal association and the availability of DIN (N€asholm et al.,2009). Our results, based on the natural d15N partitioningmethod, emphasize the importance of DON for moss N nutri-tion and add to the knowledge of mosses’ utilization of organicN sources (Forsum et al., 2006).
However, the strategies of moss DON utilization differedbetween atm-DON and soil-DON. For atmospheric N sources,DON was deposited onto moss layers with atm-NHþ
4 . Thereby,the assimilation occurred when atm-NHþ
4 could not meet mossN demand. However, the absorption of soil-DON may be morecomplex because the bioavailability of N deposition and the com-petition for soil N with soil microbes must be considered. Usingthe 15N-labeling method, Harrison et al. (2007) found that
Fig. 4 Correlation between the preference for atmospherically derivedNHþ
4 over atmospherically derived dissolved organic N (DON) (batm-NH4+;Eqn 4) and the preference for soil-NH4
+ over soil-DON (bsoil-NH4+; Eqn 5)in mosses on soils (including epilithic mosses with soil, and terricolousmosses in open fields and on floors of pine forests) in the Guiyang area.
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New Phytologist� 2013 New Phytologist TrustNew Phytologist (2013) 199: 407–419
coexisting plants of a temperate grassland showed a consistentpreference for soil DIN over soil DON and for simple (glycineand serine) over more complex amino acids (phenylalanine), butcompetition between plants and soil microbes can complicate thepicture and cause differences over time. For mosses, the motiva-tion of using soil-DON might be complex: the supply of depos-ited N sources should at least be considered. In addition, morestudies are needed to assess the explicit d15N compositions ofamino acids, and bioavailable and residual fractions of DON toexamine the relative availability of N deposition and soil Nsources to mosses. By estimating the preference for NHþ
4 overDON, a difference was found between atm-NHþ
4 and soil-NHþ4 .
Fig. 3(a) shows that mosses preferred NHþ4 to DON when assim-
ilating atmospheric N sources (0 < batm-NH4+), no matter howhigh or low the availability of soil N sources was. As explainedfor mosses on bare rock, this fact reflected the dominance ofNHþ
4 in N deposition and a strategy to avoid tissue NHþ4 accu-
mulation from continuous atm-NHþ4 inputs. Consequently, a
greater preference for NHþ4 occurred for mosses under higher
NHþ4 deposition in urban areas than in rural areas (Fig. 3a).
However, mosses showed no consistent preference for NHþ4 in
using soil N sources (Fig. 3b), but exhibited an opposite patternof bsoil-NH4+ to the batm-NH4+ (Fig. 4) associated primarily withmoss N demand and overall N availability. In urban areas, higherNHþ
4 supply from deposition and higher soil-DON availabilitycaused greater preferences for atm-NHþ
4 and soil-DON (Fig. 3).In rural areas, lower atm-NHþ
4 supply and lower soil-DON avail-ability caused a lower preference for atm-NHþ
4 but a greater pref-erence for soil-NHþ
4 .
Uncertainties and future work
Isotopic mass balance calculations in this work were based onnegligible isotopic fractionation and preference during moss Nuptake, and negligible NO�
3 assimilation in mosses under highsupply of RDN. However, the isotopic variations of plant N pooland N sources are factors that might influence isotopic partition-ing of plant N sources. For mosses, it is difficult to define thetime-scale (or temporal stability) of recorded moss d15N and theinfluence of short-term N assimilation on moss d15N. For Nsources, although particulate N in deposition might not entermoss tissues without the help of rainwater, differences might existbetween the d15N of wet N deposition and the d15N of usable Nin total deposition (dry and wet deposition). Moreover, the mea-sured DON was actually a salt-extractable fraction passedthrough a GF/F filter, although it might represent an importantfraction of the whole DON pool. The DON includes complexcompounds with different molecular weights, and presumablydifferent bioavailabilities (Knapp et al., 2005). Yet the explicitfraction and exact d15N values of moss-assimilated DON in bulkDON have not been characterized to date. Furthermore, explicitd15N signatures in N deposition have not been measured acrossall moss habitats in our study. The deposition data collected inthe urban area suggest that the d15N values of each N source aretemporarily variable to a great degree. Therefore, their mean val-ues were taken as end members of source N in the whole study
area. All above-mentioned assumptions and possible d15Nvariations in mosses and N sources need further verification infuture studies using stable isotopes to constrain moss Nutilization.
Conclusions
This study has advanced the application of the nondisturbing15N natural abundance method for interpreting plant N utiliza-tion in natural environments. Ammonium was confirmed as themain form of N species assimilated by mosses, showing higherdependence on and greater preference for atm-NHþ
4 . However,DON was revealed as an important N source, with a mean con-tribution of 38%, especially in mosses with no N available in sub-strates and in mosses receiving lower NHþ
4 deposition. Onaverage, 37% of N in mosses was derived from soil, which isgreater than estimations based on 15N-labeling of inorganic N.These results do not support the idea of exclusive reliance ofmosses on atmospheric DIN deposition, and underscore theimportance of considering DON and soil N utilization inestimating N sequestration and N deposition impacts in mossecosystems. Moreover, the contributions of DON and soil Nsources decreased with that of atm-NHþ
4 , which was related toNHþ
4 preference associated with high anthropogenic NHþ4 depo-
sition. Furthermore, terricolous mosses were found to preferatm-NHþ
4 over atm-DON, and to prefer soil-DON over soil-NHþ
4 when NHþ4 availability was elevated in deposits and soil.
The elucidation of these mechanisms provides new insights intomoss N strategies pursued in response to anthropogenic N pollu-tion, especially in regions with NHþ
4 -dominated N deposition.
Acknowledgements
We thank Prof. Janice Glime for helpful comments on the initialdraft of this manuscript. We also thank Dr Hongwei Xiao forcollecting rainwater, and Drs Haifeng Fan, Takebayashi Yu, Fu-jun Yue and Liran Bao for help with experiments. This work wassupported by a Grant for Projects for the Protection, Preservationand Restoration of Cultural Properties in Japan by the SumitomoFoundation, Grants-in-Aid for Creative Scientific Research (No.21310008), the Program to Create an Independent ResearchEnvironment for Young Researchers from the Ministry of Educa-tion, Culture, Sports, Science and Technology, Japan, and theNEXT Program (GS008) from the Japan Society for the Promo-tion of Science (JSPS). X-Y.L. was also supported by the NationalNatural Science Foundation of China (nos. 40903012,41021062 and 41273026) and the JSPS postdoctoral programfor foreign researchers (no. 09F09316).
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Supporting Information
Additional supporting information may be found in the onlineversion of this article.
Fig. S1 Map showing the location of Guiyang area and samplingsites.
Fig. S2 Contributions of soil-RDN to moss bulk N in the Guiy-ang area.
Fig. S3 Contributions of NHþ4 and DON to moss bulk N in the
Guiyang area.
Fig. S4 Soil N availability versus soil N contribution in moss Nin the Guiyang area.
Table S1 Level and d15N of dissolved N in wet deposition atGuiyang, southwestern China
Table S2 Bulk N and d15N in natural mosses in the Guiyangarea
Notes S1 Additional methodological details for the study area,sampling and isotopic analyses.
New Phytologist (2013) 199: 407–419 � 2013 The Authors
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Notes S2 Additional results for characteristics of soils undermosses.
Notes S3 Additional results of isotopic mass-balance calculationsbased on RDN.
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