Spatial patterns nitrogen transfer models of ectomycorrhizal networks in a Mongolian scotch pine plantation

Yanbin Liu•Hongmei Chen•Pu Mou

Introduction

Mycorrhizal mycelial networks link plants to facilitate fungal colonization and/or the transfer of compounds among plants(Leake et al.2004;Heijden and Horton 2009;Barto et al.2011;Simard et al.2012;Albarracín et al.2013).Two types of common mycorrhizal networks(CMNs)have been identi fi ed based on the type of mycorrhizae involved:arbuscular endomycorrhizal(AM)network and ectomycorrhizal mycorrhizal(EM)network(Selosse et al.2006).EM networks are mostly studied on their functions of providing a variety of services to plants and ecosystems including nutrient uptake and transfer(He et al.2003,2005,2007;Moyer-Henry et al.2006;Correˆa et al.2008),seedling support(McGuire 2007;Teste et al.2009),prevention of nutrient leaching(Heijden and Horton 2009),internal cycling of nutrients(Callesen et al.2013),and plantcompetition (Booth 2004;Barto etal.2011,2012).

Many temperate and mediterranean woody species form EM networks with basidiomycete and ascomycete fungi(Selosse et al.2006).Using network analysis,Bahram et al.(2014)concluded that certain ectomycorrhizal communities displayed modularity and attributed to partner selectivity and consequent context dependent.They also showed that the EMs exhibited non-nested or anti-nested patterns,contrasting to other mutualistic interactions.

Nitrogen is often transferred among plants through EM networks(Selosse et al.2006;Teste et al.2009).Recent studies have focused on nitrogen uptake and transfer from N2- fi xing donors to non-N2- fi xing receivers to demonstrate mutualistic functions among plants via mycorrhizal networks(He et al.2005,2007;Moyer-Henry et al.2006).However,the net N transfer in EM networks and the spatial distribution of EM networks were not found to be ef fi cient in most of the studies.

There are few tools to evaluate ectomycorrhizal roles in N transfer in situ.Analyses of natural abundance N isotope ratios(15N:14N expressed as δ15N relative to standard),as an integrator of N transfer,can provide a glimpse into mycorrhizal functional ecology within soil pro fi les and across biomes(Dawson et al.2002;Fry 2006;Nave et al.2013;Hobbie et al.2014;Pena and Polle 2014;Mayor et al.2015a,b).In this study,we examined N transfer among trees via the EM network using a stable isotope15N approach in a monoculture tree plantation and characterized the spatial patterns of the EM networks to increase our understanding of the structure and function of EM networks in ecosystems,which may lead to a deeper understanding of ecological stability and evolution and thus new theoretical approaches to improve conservation practices for the management of the Earth’s ecosystems.

We injected15N solution into the soil at selected locations and periodically sampled the leaves of the trees deviating from the injection locations.We tested the following hypotheses by analyzing changes and variation in15N concentrations in leaves:(1)EM networks in this plantation were structured such that they transfer N homogeneously if leaf15N concentration varies in a similar manner;otherwise,local EM networks would be con fi rmed and functionally differ.(2)EM networks transfer N at high ef fi ciency if there is a strong distance-dependent reduction in leaf15N.

Materials and methods

Study site

The study was conducted in a Mongolian Scotch pine(Pinus sylvestrisvar.mongolicaLitv.)plantation in the Xiaoxing’anling Mountain,nearWuying Township,Yichun City, Heilongjiang Province, China(48°06′11.54′N,129°15′03.58′E).This area is mountainous with mild slopes and lower-elevation reliefs.The mean annual precipitation is 637.0 mm (1958–2006,49 a)(Cheng et al.2010),mostly concentrated as rainfall during the growing season(June to August),and winter snow depth can be up to 113.8 mm.The snow comprises about 17.9% of the annul precipitation.The monthly mean temperature ranges from-23.5 °C in January to 20.3 °C in July.The lowest temperature was-44.9°C in 1970,and the highest was 37.5°C in 2010.The average annual frostfree period lasts 111 days.The regional vegetation is a temperate mixed conifer–hardwood forest dominated by Korean pine(Pinus koreiensisSieb.et Zucc.),Korean spruce (PiceakoraiensisNakai),needle fi r(Abies nephrolepisMaxim.),white birch(Betula platyphyllaSuk.),Manchurian ash(Fraxinus mandschuricaRupr.),Chinese corktree(Phellodendron amurenseRupr.)and Chinese walnut(Juglans mandshuricaMaxim.).The local soil is dark-brown with rich organic matter concentrations,equivalent to Hap-Boric Luvisols in the US soil taxonomy.

The plantation was the result of forestation in an 18-ha construction pit abundant of coarse sand and small gravel.In 2007,the site was covered with 15 cm thick local forest top soil,and 3-yr-old seedlings of Mongolian Scotch pine were planted with spacing of 2×3 m.On average,the plant-available N in the top soil was 100±5 mg kg-1.At the time of the study,the trees were 2–3 m tall,7–8 years old with DBH of 4–6 cm.The canopy was not closed yet.Understory vegetation was scattered with low total cover,which included forbs,native perennial grasses,mainlyArtemisiaspp.,Carexspp.,andAchilleaspp.,and 98% of pine roots occurred in the top 30 cm of soil.

The top soil(0–30 cm surface layer)throughout the experimental site was full of dense white rhizomorphs,mainlySuillus luteus(L.:Fr.)Grey.based on sporocarp inventory and DNA sequencing(Liu’s unpublished data),Gomphidius rutilus(Schaeff.:Fr.)Land.Et Nannf.,Laccaria laccata(Scop.:Fr.)Berk.et Br.andLycoperdon pyriformeSchaeff.:Pers.scattered in the fi eld at low abundance.Some other saprophytic fungi,includingHygrophorus conicus(Fr.)Fr.,H.agathosmus(Fr.)Epicr.,Marasmius androsaceus(L.:Fr.)Fr.,Phallus tenuis(Fisch.)D.Ktze.,andCoprinus parouillardiQuél.were also observed.

15N application,leaf sampling and measurements

In August 2011,four plots(20×20 m)were randomly selected,which were far enough away from each other and separated by cement tracks.As a result,they did not interfere with each other.At the center of each plot,125 ml 5 at.% 0.15 mol/Lsolution wasinjected within a 2.5-cm radius.To avoid slope and precipitation interference,four sample lines expanded from plot center to four directions and were as perpendicular as possible according to the seedling pattern,slope and orientation.Each sample line was 14–15 m and contained 5–6 pines.In total,we sampled 20 pines,21 pines,20 pines and 20 pines in four respective plots.

At 0,2,6,30 and 215 days after15N application,we sampled needles(current year)of each pine along a transect line.Needles were immediately sealed in Ziplock plastic bags and stored in cool boxes until taken to the lab.Needles were oven-dried at 65°C for 48 h,ground and sieved through a 0.2 mm sieve.

Needle δ15N values and N concentration(in%)were measured on 10±1 mg of each sample at the Stable Isotope Laboratory in the Chinese Academy of Forestry(Beijing,China)with an elemental analyzer(Flash EA1112 HT,Thermo Fisher Scienti fi c,USA)coupled with a gas isotope ratio mass spectrometer(DELTA V Advantage,Thermo Fisher Scienti fi c).δ15N(‰)were calculated as:δ15N(‰)=[(Rsample/Rstandard)-1]× 1000,whereRis the ratio of15N/14N of the sample and standard(Knowles and Blackburn 1993).

Statistical analyses

Data from four plots were pooled together and divided into fi ve groups according to the distance of the sampled tree from the15N application point,i.e.,1–3,＞3–5,＞5–7,＞7–9,and ＞9–11 m.All data were analyzed with a Kolomogrov–Smirnov goodness of fi t test and Levine’s test to examine their normality and homogeneity of variance.When the data were not normally distributed,data were transformed to meet the requirements.A one-way ANOVA was used to compare means N and15N concentrations among different groups,and Turkey’s honestly signi fi cant difference(HSD)test was used to test for differences among group means if ANOVA results were signi fi cant(P＜0.05).If the data,even after transformation,did not satisfy the requirement of normality and homescedasticitic tests,Kruskal–Wallis test was used to compare the fi ve group means.To assess the relationship between groups of variables,Spearman’s rank correlation test was performed on all data.We used linear regression models to examine the relationship between N and15N concentrations of the needles and the relationship between the needle15N concentrations and the distance away from the injection points.Time interval and excess meant the needle N and15N concentrations of the same tree from the day 0 to day 2,day 2 to day 6,and day 6 to day 30,and day 30 to day 215.We wanted to determine any changes in needle N and15N concentrations among time intervals.All statistical analyses were performed using SPSS(v.21;SPSS,Inc.,IBM,Armonk,NY,USA)and Origin(OriginPro v.9.1.0,OriginLab Corp.,Northampton,MA,USA)for Windows,and all differences were considered signi fi cant atP＜0.05.

Results

Needle N concentration and15N/14N ratio pattern

NeedleNconcentrationand15N/14Nratiorangedfrom1.07to 2.77%,and from 0.36648 to 0.37669,respectively.ANOVA resultsdidnottestforsigni fi cantdifferencesamongthemeans ofneedleNconcentrationsorneedle15N/14Nratioamongthe four plots(P%N=0.06,=0.88).The needle N and15N/14Nratio(n=227)increasedsigni fi cantlyafter30 days,up to 31 and 0.42%,respectively(Fig.1a,c).The needle N concentration was highest on day 30 after the treatment and the15N/14N ratio was highest on day 215.The needle N concentrationsincreasedby20,18,31and23%onday2,6,30 and 215 after treatment,respectively.The15N/14N ratio increased by 0.09,0.17,0.35 and 0.42%,on day 2,6,30 and 215 after treatment,respectively.The needle N concentration excess,aswellas15N/14Nratioexcesssigni fi cantlydecreased over time(Fig.1b,d).

Variations and correlations between needle N concentration and15N/14N ratio

Needle N concentration and15N/14N ratio were not positively correlated through time (R2=0.40,n=5,P=0.156;Fig.2a).Needle N concentration excess and15N/14N ratio excess were positively correlated across different time intervals(R2=0.89,n=4,P＜0.05;Fig.2b).

Needle15N/14N spatiotemporal pattern

Needle15N/14N ratio increased with time,but was not signi fi cantly correlated with distance(Figs.1c,3).There was weak trend of decreasing15N/14N ratio with increasing distance at day 0,day 6 and 30,but the ratio increased with increasing distance at day 2.

Fig.1 Changes in pine needle N and15N concentrations in a Mongolian Scotch pine plantation:a N concentration,b time interval N concentration change,c15N/14N ratio,d time interval15N/14N ratio change.Values are%and at.%excess(n=227–232)with SE bars.Values with different letters indicate signi fi cant differences among means according to Tukey’s honestly signi fi cant difference tests(P＜0.05)

Fig.2 Relationship between N concentration and15N/14N ratio of pine needle in a Mongolian Scotch pine plantation.a N concentration and15N/14N ratio over time,b N concentration excess and15N/14N ratio excess at different time intervals.Values are mean excess(n=4–5)with SE bars

Variations and correlations between needle N%,δ 15N and15N/14N contents in distance

At 1–3 m,pine needle δ15N was negatively correlated with that at 5–7,7–9 and 9–11 m;at 3–5 m,δ15N was also negatively correlated with that at 5–7,7–9,and 9–11 m,then they were not correlated.At 5–7 m,pine needle δ15N was a few signi fi cant positively correlated with that at 7–9 m,and that at 7–9 m with that at 9–11 m(P＜0.05)(Table 1A).At 3–5 m,pineneedleNcontentwasnegativelycorrelatedwith 1–3 and 7–9 m,N at 9–11 and 7–9 m was negatively correlated;and the rest were positive correlations,then they were not correlated(Table 1B).For needle15N/14N content,signi fi cant positive correlations were found among all the distance groups(Kruskal–Wallis ANOVA,P＜0.001 orP＜0.01),but not between groups(one-way ANOVA,NS);forδ15NandN,thecorrelationwassigni fi cantamonggroups(one-way ANOVA,P＜0.001 orP＜0.01),some within the group were signi fi cant(δ15N of 1–3 and 3–5 m atP＜0.001;%N of 5–7 m atP＜0.01),some intragroup correlations were not(Table 2).

Fig.3 Correlations between needle15N/14N ratios and distance from the injection point in a Mongolian Scotch pine plantation.The regressions at different times are:day 0,15N/14N(at.%)=0.36661+(-1.05E-5)×Distance(m),adjusted r2=0.05,P=0.22;day 2,15N/14N(at.%)=0.36685+(1.44E-5)×Distance(m),adjusted r2=0.08, P＜0.05; day 6, 15N/14N (at.%)=0.36726+(-8.76E-6)×Distance(m),adjusted r2=0.15,P＜0.0001;day 30, 15N/14N (at.%)=0.36766+(-2.35E-5)×Distance (m),adjusted r2=0.08,P＜0.05

Discussion

Spatiotemporal patterns in EM networks

EM networks are complex adaptive systems(Nave et al.2013),which have been modelled as adaptive dynamic networks of interacting parts where feedback and crossscale interactions lead to self-organization and emergent properties (Beiler etal.2010).Understanding the architecture of the EM networks in the fi eld(e.g.,the physical components,the spatial extent and their relationships)is a prerequisite to understanding how EM networks function and how they affect plant populations,communities,and dynamics in forests(Selosse et al.2006;Simard et al.2012).In this study,we showed that the stable isotope15N rapidly spread rather far away from the injection spots and appeared in the tree needles within 2 days(Fig.1)throughout the four study plots.This result indicated that effective EM networks were ubiquitous in this study plantation and might have a rather uniform distribution.Nitrogen and carbon are thought to travel through EM networks together as simple amino acids(Simard et al.2015).These molecules are transferred through the EM network rapidly,from donor plants to the fungal mycelium within 1 or 2 days and to the shoots of neighboring plants within 3 days(Wu et al.2002;Heaton et al.2012).The high dissimilarity of fungal assemblages at roots of the same genotypes at spatial distances of some meters was unexpected because the overall similarities of the fungal communities in the soil cores of the plot did not differ signi fi cantly;thereby,asymmetric competition between conspeci fi c neighbors can be avoided(Lang et al.2013).But Toju et al.(2016)reported that diverse root-associated fungi could coexist in highly compartmentalized networks within host roots and that the structure of the fungal symbiont communities could be partitioned into semi-discrete types even within a single host plant population.The largely uncorrelated relationships between the needle15N concentrations and distance to the injection points(Fig.3)indicated a rapid15N transfer with the networks.The accelerated increases of needle15N contents as thesampling period increased suggested the existence of longlasting effective EM networks in this Mongolian Scotch pine plantation(Fig.1).

Table 1 Spearmen’s rank correlation coef fi cient(ρ)for pine needle δ 15N(‰)(A),N(%)(B),and15N/14N(at.%)(C)among distance groups in a Mongolian Scotch pine plantation

Table 2 Effect of distance from N-loading points on needle N,δ15N and15N/14N ratios by Kruskal–Wallis ANOVA intragroup and one-way ANOVA intergroup in a Mongolian Scotch pine plantation

Nitrogen transfer models

He et al.(2005)showed that nitrogen transfer was enhanced by mycorrhiza formation and that transfer rates were greatest in the mycorrhizal treatment.Our results demonstrated a rapid transfer of nitrogen through the EM networks to the pine tree,but the amount of nitrogen transferred was rather small,as indicated by the increments of15N in fractions of a percentage.Does this result mean that the CMNs are not effective in nitrogen transfer?We consider that more evidence is still needed;the few studies on interplant transfer of nutrients through CMNs focused mainly on transfer from N2- fi xing plants to non fi xing ones at more local scales(He et al.2005;Moyer-Henry et al.2006).Our data might be the result of several factors:(1)A dilution effect as the element spread from the injection points;however,the weak correlations between needle15N contents and distance(Fig.3)did not support such an effect.(2)Nutrient transfer capability of CMNs may be limited;however,the needle15N content in some of the closer trees(1–3 m)was very high.Some may argue that there might be another channel to transfer15N to these trees,but we do not have data for or against this idea,and more studies are needed.(3)High turnover rates in the tree-CMN connection may disturb the N transfer function of CMN.Ectomycorrhizal hyphae turnover is estimated at 46 days,rhizomorphs at 11 months,and EM root tips from 1 year to 6 years(Bledsoe et al.2014).(4)Needles in N-loaded plots became enriched in15N,re fl ecting decreased N retention by mycorrhizal fungi and isotopic discrimination against15N during loss of N.Needles in N-limited(control)plots became depleted in15N,re fl ecting high retention of15N by mycorrhizal fungi(Ho gberg et al.2011).Stronger15N retention of ectomycorrhizal fungi resulted in a consequently transfer of15N-depleted N to their tree hosts(Ho gberg et al.1999;Hobbie and Colpaert 2003;Hobbie et al.2008;Mayor et al.2012).(5)Nitrogen immobilization from soil organisms may also affect the transfer effectiveness of N and15N.Net N transfer was much greater when N was supplied as15NH4+than15NO3-(He et al.2005).Kranabetter et al.(2015)found ammonium uptake was greatest in the spring at medium-N and rich-N sites and averaged over 190 nmol m-2s-1forTomentellaspecies, and nitrate uptake was only 8.3 nmol m-2s-1.The cation NH4+is bound to negatively charged sites on clay lattices in soil,reducing mobility and leading to reduced availability(Brady and Weil 2002).Nitrogen additions led to expected increases in foliar N/P ratios,reductions in δ15Nfungi-plantvalues,and15N enrichment of soil nitrate(Mayor et al.2015a,b).None of these potential factors could be ruled out by the unexpected results of our study,which raised more questions to examine in future research.

Application of network theory to potential EM networks

Network theory provides a useful framework for describing the structure,function and ecology of EM networks(Southworth et al.2005;Selosse et al.2006;Beiler et al.2015).Southworth et al.(2005)viewed trees as nodes and fungi as links(the so-called phytocentric perspective)and considered that the distribution of potential mycorrhizal links was random with a short tail,implying that all the individuals trees are more or less equal in linking fungi into a potential network.However,from a mycocentric point of view that fungi are nodes and trees are links,certain fungus may act as hubs with frequent connections to the network.Our study supports the phytocentric point;the ECM network was not patchily distributed(Tables 1,2;Figs.1 and 3),but ubiquitous and might be evenly distributed.This fi nding indicates that CMNs are random networks and that all nodes have the same probability of being attached to a link.Our data revealed CMNs were random networks,though rather indirectly,through signi fi cant interdistance correlations of needle15N contents,but insigni fi cant needle15N content differences among distance groups(Tables 1,2).Pickles et al.(2012)reported similar results.Beiler et al.(2010)found that most trees in a multicohort old-growth forest were linked in a scale-free EM network,where large trees served as hubs.Beiler et al.(2015)also found that large mature trees acted as network hubs with a signi ficantly higher node degree compared with smaller trees in Douglas- fi r forests.

Conclusions

In natural ecosystem,resource transfers through EM networks are highly complex,the networked fungi and plants interact to govern the magnitude,direction,fate and consequences of resource transfers,which have important consequences for plant communities and may in fl uence plant establishment or growth,intra-and interspeci fi c competition or facilitation,and stand dynamics and succession(Nara 2006;Simard et al.2012,2015;Koide et al.2014).Tracing studies based on15N external labelling and15N natural abundance techniques consistently have found that the direction and magnitude of N transfer is from N2-fi xing,N-fertilized or N-enriched source plants to non-N2fi xing,unfertilized or N-depleted sink plants(He et al.2005,2007;Moyer-Henry et al.2006);however,the net N transfer in EM networks and the spatial distribution of EM networks were not found to be deterministic in most of the studies.We used stable isotope15N labeling method to study the EM networks in a monoculture pine plantation and characterize the spatial patterns of the networks and N transfer among the trees via the network.We concluded that EM networks were ubiquitous and uniformly distributed in the Mongolian pine plantation,the N transfer ef fi ciency was very high and N fractionation was found.Deeply understanding the N transfer model and spatial pattern is important not only analyzing N dynamics and distribution in N-limited ecosystems,but also the role of N in regulating N and C transfers through networks.Because the potential bene fi ts of N transfer mediated by EM networks are great in agricultural and forest systems,more research is warranted on this type of N transfer in the fi eld.

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