Adaptational significance of variations in DNA methylation

Adaptational signi?cance of variations in DNA methylation

in clonal plant Hierochloe glabra(Poaceae)

in heterogeneous habitats

Rujin Bian A,Dandan Nie A,B,Fu Xing A,D,Xiaoling Zhou A,Ying Gao A,

Zhenjian Bai A and Bao Liu C

A Institute of Grassland Science,Key Laboratory of Vegetation Ecology,Ministry of Education,

Northeast Normal University,Changchun130024,China.

B Hunchun Entry–Exit Inspection and Quarantine Bureau,Export Processing Zone,Hunchun133300,China.

C Key Laboratory of Molecular Epigenetics of Ministry of Education and Institute of Genetics&Cytology,

Northeast Normal University,Changchun130024,China.

D Corresponding author.Email:xingf522@https://www.360docs.net/doc/c337409.html,

Abstract.As a prominent epigenetic modi?cation,cytosine methylation may play a critical role in the adaptation of plants to different environments.The present study sought to investigate possible impacts of differential levels of nitrogen(N) supply on cytosine-methylation levels of a clonal plant,Hierochloe glabra Trin.(Poaceae).For this purpose,nitrate was applied at concentrations of0,0.15,0.30and0.45g N kg–1soil,and ecologically important morphological traits were measured.The methylation-sensitive ampli?cation polymorphism method was also conducted to analyse the variations in DNA cytosine methylation.Our results showed that N addition reduced CHG cytosine-methylation levels markedly compared with control plants growing in homogeneous pots(P=0.026).No substantial differences were observed in morphological traits at the end of the growing stage,except for the highest ratio of leaf area to leaf dry mass in the medium-N patch(P=0.008).However,signi?cant linear regression relationships were found between cytosine-methylation levels and morphological traits,such as bud number and rhizome length and biomass.In conclusion,the higher cytosine-methylation level may activate asexual reproduction to produce more offspring and expand plant populations,possibly helping clonal plants to adapt to heterogeneous habitats.

Additional keywords:epigenetic variation,methylation-sensitive ampli?ed polymorphism(MSAP),nutrient heterogeneity, phenotypic plasticity.

Received23June2012,accepted10April2013,published online24May2013

Introduction

It is generally believed that genetic variation is the sole basis for morphological diversity among organisms(Linhart and Grant 1996;Herrera and Bazaga2009).Nevertheless,some researchers have challenged this view and suggested that the phenotypic plasticity of plants cannot be controlled simply by genetic variation because of low nucleotide mutation rates(Boyko and Kovalchuk2011).Meanwhile,several recent studies have demonstrated that the alteration of epigenetic modi?cations could affect the phenotypic characters of some species,allowing individuals to better cope with different environmental challenges,without changes to DNA sequences(Kalisz and Purugganan2004;Jablonka and Raz2009;Lira-Medeiros et al.2010).As one of the most important epigenetic modi?cations,cytosine methylation has been shown to be not only inheritable per se,but it may also affect heritable alterations in gene expression and,ultimately,contribute to phenotypic diversity(Verhoeven et al.2010a;Grativol et al.2012). Researchers have provided considerable evidence that asexual populations or clones generated by vegetative propagation could change morphological traits by altering epigenetic modi?cations in response to changing environments(Fang and Chao2007; Monteuuis et al.2008;Richards2011).

In natural ecosystems,habitat heterogeneity is a common environmental feature,and plants have developed various sophisticated mechanisms to cope with patchy resource distribution(Farley and Fitter1999;Hodge2006).In particular,clonal plants have constituted morphological and physiological plasticity as a strategy for more ef?cient resource use,to guarantee their survival under adverse conditions (Welham et al.2002;Day et al.2003).However,most studies have investigated only the anatomical or morphological responses to resource heterogeneity and neglected plant molecular changes.

CSIRO PUBLISHING

Australian Journal of Botany,2013,61,274–282

https://www.360docs.net/doc/c337409.html,/10.1071/BT12242

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Nitrogen has been reported as an important factor in the activation of new rhizomes(Hutchings and de Kroon1994)as well as in increasing ramet number and biomass in clonal plants (Bai et al.2009).Indeed,application of N fertiliser is a common management measure to maintain nutrient balance in some grassland ecosystems(Drake et al.1963).In China,grassland fertilisation is still at the small-scale experimental stage,and soil types vary spatially and temporally in the natural environment (Ni and Zhang2000).Studies focussed on phenotypic plastic responses and foraging behaviours under heterogeneous habitats have suggested that clonal plants can be more sensitive and responsive to adapt to varying conditions(Dong1996;Gao et al.2012).However,the responses of cytosine-methylation variations of clonal plants to different levels of N supply remain unclear.

In the present study,we selected a perennial herb species Hierochloe glabra Trin.as our experimental subject.The species is a rhizomatous clonal plant that is widely distributed in the meadow steppe of the eastern Eurasian steppe zone,where nutrient heterogeneity is a ubiquitous feature of the natural habitat.We designed a multi-gradient N-supply experiment to evaluate how H.glabra responds to heterogeneous nutrition, and try to explain the adaptational mechanisms in the barren meadow steppe by detecting the relationships between epigenetic alteration and plant phenotypic variation.

Materials and methods

Plant material and study site

Hierochloe glabra is a perennial herbaceous species that is widely distributed over the Songnen Plain of north-eastern China.This species usually occupies unfertile castanozem and saline-alkalised soils and reproduces both sexually through seed germination and asexually through vegetative propagation of rhizomes(Wang and Ba2008).To ensure that ramets selected in our experiment had the same genetic base,a single ramet of H.glabra collected from natural grassland was transplanted into a cube-shaped nursery pool to propagate new ramets before we started our experiment.At the bottom and four sides of the pool, we used cement to avoid inside or outside growth of roots.

The experiment was conducted at the Songnen Grassland Ecological Research Station of North-east Normal University, Jilin Province,PR China(44 450N,123 450E).The study area has a semiarid continental climate,with an annual precipitation of 350–450mm,and the annual mean temperature is4.6–6.4 C (Gao et al.2008).

Experimental design and growth conditions

An open-ended greenhouse experiment was conducted to assess the impact of different N concentrations on the clonal growth of H.glabra.Sixty plastic pots(60cm in diameter and20cm in depth)were placed randomly,to homogenise the potential effects of photoperiod and temperature.Every plastic pot was divided into four equal compartments by plastic partitions,with a space (0.1?0.1m2)left in the central area for the transplantation of clones.The plastic partitions were?rmly attached to the sides and bottoms of pots using plastisol,so as to prevent nutrients from the four compartments from mixing.A circular hole(2cm in diameter)at the bottom of each compartment was covered with window screening to ensure aeration and to prevent rhizomes from growing out of the hole.Each of the compartments was ?lled with8kg of soil taken from a natural grassland near the

ecological research station.The soil was passed through a4-mm sieve to remove plant litter.In early May2010,60H.glabra ramets of a uniform size from our nursery pool were transplanted individually into the central areas of the pots.These selected ramets were descended from one parent ramet and had the same genetic base.There were35heterogeneous pots,with the four compartments de?ned sequentially as no N(NN),low N(LN), medium N(MN)and high N(HN;Fig.1).Here,we considered that because of resource translocation in heterogeneous patches through interconnected rhizomes,ramets in the NN patch might be affected by N.Therefore,we designed another25 homogeneous pots to serve as controls;these pots were partitioned in the same manner as above,but no N was added. In the following analysis,CK represented the average of values measured in the homogeneous pots.Patches in the heterogeneous pots were fertilised twice with equipotent ammonium nitrate solution,and the concentrations of added N were0for NN, 0.15for LN,0.30for MN and0.45g N kg–1soil for HN.The same volume of water was added to the control plants in homogeneous pots.During the growing season,all pots were rotated three times to prevent the possible effects of orientation. All plants were watered when necessary.

Measurements and data analysis

We reserved120g of soil for N analysis at the beginning of the experiment,and two soil samples of each patch from the heterogeneous pots were also collected in the middle of the growing season(4August).All soil samples were stored at room temperature until processed.Soil N was determined by the Kjeldahl method(Bremner and Mulvaney1982),and pH was measured in a1:5soil–water suspension after shaking for 30min(Walkley and Black1934).Concentrations of available N are expressed as mg kg–1of dry soil mass.The values for soil-available N are the average of the two samples.

At the conclusion of the experiment(2September),the number and height of offspring ramets in each patch were recorded.Fifteen young,fully expanded leaves of ramets in

NN LN

HN MN

Fig.1.Schematic picture of the designed heterogeneous patches,NN,LN, MN and HN,in our experiment,with nitrate added at concentrations of0, 0.15,0.30and0.45g N kg–1soil,respectively.Homogeneous pots without added nitrogen were also divided into four patches.Clones of Hierochloe glabra were transplanted in the central area,giving each patch an a priori equal chance of being colonised.

Habitat adaptation through methylation variations Australian Journal of Botany275

each patch were randomly collected from the stems,and the leaf area was measured.Samples were then oven-dried at60 C for at least2days and the dry mass was determined.We de?ned the ratio of fresh leaf area to dry mass as the speci?c leaf area(SLA; Gower et al.1999).Then,the aboveground parts of all ramets in the four patches were cut at the soil surface,packed into envelopes,dried to a constant mass and weighed.The underground parts in each patch were dug up,washed and then separated into buds,rhizomes and?brous roots.The number of buds was counted.The spacer length(rhizome length between two adjacent ramets)and the absolute length of all rhizomes in each patch were measured.The underground parts were also dried at60 C to determine the underground biomass.

Prior to analyses,all variables were tested for normality and homoscedasticity of variance.One-way ANOVA was used to determine whether heterogeneity induced a signi?cant difference in the measured indices and in the level of DNA cytosine methylation,and a linear regression between the phenotypic indices and methylation levels was applied with SPSS software(v.17.0for Mac;SPPS Inc.,Chicago,IL,USA).A multiple-comparison analysis was performed using Duncan’s multiple-range test at the signi?cance level of a=0.05. Sample collection and DNA extraction

We reserved?ve heterogeneous and homogeneous pots in advance for DNA extraction.Five leaf-tissue samples from each patch were randomly collected.The leaves were scrubbed softly with C2H5OH–H2O(3:1,v/v),packed with tinfoil to prevent contamination,and then kept atà80 C for the following extraction of DNA.Genomic DNA was isolated by using an improved CTAB method and puri?ed with phenol extractions(Kidwell and Osborn1992).Ultimately,three DNA samples from each patch were used as starting material for the methylation analysis described later.

Methylation-sensitive ampli?ed polymorphism analysis Alterations in cytosine modi?cation of H.glabra were detected using methylation-sensitive ampli?ed polymorphism(MSAP). The MSAP method is a modi?cation of the standard ampli?ed fragment length polymorphism technique that incorporates a pair of isoschizomers,Hpa II–Msp I,which recognise the same tetranucleotide50-CCGG but have differential sensitivity to methylation modi?cations of the two cytosines(Reyna-López et al.1997;Cervera et al.2002).Hpa II is inactive if one or both cytosines are methylated at both DNA strands,but it cleaves when one or both cytosines are methylated in only one strand. Msp I,in contrast,cleaves C5m CGG but not5m CCGG (McClelland et al.1994).For clarity,we hereby de?ne these two major types of modi?cations of cytosine methylation as CG methylation(a band present in Msp I but not in Hpa II digest)and CHG methylation(a band present in Hpa II but not in Msp I digest).The MSAP method consists of digestion and ligation reactions;the pre-ampli?cation and selective ampli?cation reactions and the detection reactions are detailed in Portis et al.(2004).One pair of pre-selective primers and19pairs of selective primers were used(Table1).The ampli?cation products of MSAP were loaded onto a preheated6%denaturing polyacrylamide gel(43.5?33.5cm),with electrophoresis performed at55W for3h,followed by staining of the gel. Total methylation content(%)was calculated by dividing the total number of bands scored by a CG-methylation or CHG-methylation site.Frequencies(%)of methylation polymorphism of the clones in the heterogeneous pots were calculated by dividing different variant MSAP bands into CG hypermethylation(CG+),CHG hypermethylation(CHG+),CG hypomethylation(CG–)and CHG hypomethylation(CHG–) against the total number of scored bands.

According to the presence or absence of the bands from speci?c isoschizomer digestions,samples were scored to represent the following:a fragment was(1)present in both enzyme combinations(11),(2)absent in both enzyme combinations(00)or(3)present only in either Eco RI–Hpa II (10)or Eco RI–Msp I(01)products.Condition(1)denotes a non-methylated state,condition(3)corresponds to a methylated state and condition(2)is uninformative,because it could be attributed to either fragment absence or hypermethylation (Xiong et al.1999;Ashikawa2001;Cervera et al.2002). Furthermore,we divided the ampli?ed DNA fragments into two types,namely methylation-insensitive polymorphisms (MISPs)and methylation-sensitive polymorphisms(MSPs). MISP represents the band presence for both enzyme combinations and is recorded as‘1’,with others being recorded as‘0’;MSP represents the band presence for either Eco RI–Hpa II or Eco RI–Msp I and is recorded as‘1’,with others

Table1.Sequences of adapters and primers used in methylation-sensitive ampli?cation polymorphism analysis

Adaptor or primer EcoR I(E)Hpa II–Msp I(HM)

Adaptor150-CTCGTAGACTGCGTACC-3050-GATCATGAGTCCTGCT-30

Adaptor250-AATTGGTACGCAGTC-3050-CGAGCAGGACTCATGA-30

Pre-selective primer50-GACTGCGTACCAATTCA-30(E00)50-GATGAGTCTAGAACGGF-30(HM00)

Selective primer EA(E00+AAC)HM1(HM00+TAC)

EB(E00+AAG)HM2(HM00+TAG)

EC(E00+ACA)HM3(HM00+TCT)

ED(E00+ACT)HM4(HM00+TCG)

EF(E00+ACG)HM5(HM00+TTC)

EG(E00+AGC)HM6(HM00+TTG)

EH(E00+AGG)HM7(HM00+TTA)

EI(E00+AGA)HM8(HM00+TGA)

EJ(E00+ATC)HM10(HM00+TGT)

276Australian Journal of Botany R.Bian et al.

being recorded as ‘0’.To analyse the epigenetic similarity between two pairs of samples,a dendrogram was generated by using the unweighted pair-group method using arithmetic averages (UPGMA)in NTSYS (Rohlf 2000).

A set of 27typically methylation-variant bands were isolated and re-ampli ?ed with the appropriate selective primer combinations,and then the PCR products were ligated into the pMD18-T vector (TaKaRa,Dalian,China).The cloned DNA segments were sequenced on an ABI3730(Applied Biosystems,Foster City,CA,USA).The sequences obtained were searched using the BlastN and BlastX programs on the National Center for Biotechnology Information website (https://www.360docs.net/doc/c337409.html,/,accessed 10September 2011).Results Soil inorganic N

The soil total N,available N and pH at the beginning of the experiment were 0.65g kg –1,26.30mg kg –1and 8.28,respectively.One week after the last fertilisation (26July),the concentrations of available N in NN,LN,MN and HN patches of heterogeneous pots were 48.03,126.08,261.96and 326.17mg kg –1,respectively.ANOVA analysis showed that there was a signi ?cant treatment effect on concentrations of soil inorganic N (P <0.001).

Measurement of morphologic indices

Morphologic traits of ecological importance were measured both on treated individuals from heterogeneous pots and control plants in homogeneous pots.Bud number,total biomass and rhizome length of H.glabra exhibited an obvious downward trend in contrast to the amount of N added.Ramet height decreased from NN to HN patches,and was higher in the CK patch than in the

HN patch.Ramet number in each patch showed no signi ?cant difference,and this indicated that new offspring ramets were randomly placed in the surrounding patches.No signi ?cant effect of heterogeneity was found on mean spacer https://www.360docs.net/doc/c337409.html,rge differences among the patches were found for the SLA (P =0.008),with the highest value in the MN patch (Fig.2).MSAP analyses of DNA methylation

Cytosine-methylation polymorphism in the leaves of H.glabra was clearly exhibited on the electrophoretic pro ?les (Fig.3).In total,1649reproducible fragments,ranging from 100to 500bp,were recorded.

An obvious reduction of cytosine-methylation levels induced by N addition was present in our study.Ramets growing in CK patches showed signi ?cantly higher total methylation levels (P =0.027;Fig.4a ).CG/CHG-methylation levels in NN,LN,MN and HN patches were 27.93%/11.51%,27.92%/11.49%,27.94%/11.38%and 27.63%/11.39%,respectively,and in the CK patch,they were 27.92%/11.83%.Multiple-comparison analysis by Duncan ’s test showed that the CHG-methylation level in the CK patch was signi ?cantly (P =0.026)higher than in the four treatment patches.However,these differences were not signi ?cant (P =0.556)at the CG-methylation level.

The changed methylation levels of CG and CHG included both decreases (hypo-)and increases (hyper-)in methylation,which were re ?ected by the gain and loss of bands in either or both of the enzyme digestions in MSAP.The total methylation-variation ratios of ramets in NN,LN,MN and HN patches compared with the control patch were 2.51%,2.69%,2.65%and 2.91%,respectively,and were not substantially different (P =0.474),and there was an ascending tendency in accordance with the concentration of N (Fig.4b ).Neither hypo-or hypermethylation in CG and CHG exhibited signi ?cant changes.

20151050

86420

250200

150100500

1086420

500

4003002001000

353025201510

246CK NN LN MN HN

CK NN LN MN HN CK NN LN MN HN

CK NN LN MN HN

CK NN LN MN HN CK NN LN MN HN R a m e t h e i g h t (c m )

R h i z o m e l e n g t h (c m )

S L A (c m 2/g )

T o t a l b i o m a s s (g )

S p a c e r l e n g t h (c m )

B u d n u m b e r

R a m e t n u m b e r

Fig.2.Responses of the morphological indices of Hierochloe glabra to heterogeneous habitat patches (NN,LN,MN and HN,with nitrate added at concentrations of 0,0.15,0.30and 0.45g N kg –1soil,respectively)and to homogeneous habitat (CK).Data are the means ?s.e.m.Columns with different letters differ signi ?cantly (P <0.05).

Habitat adaptation through methylation variations Australian Journal of Botany 277

Cluster analysis

UPGMA cluster analysis of MSP (Fig.5a )showed that most of the individuals from the same patch clustered together,whereas three samples from the NN patch were far from each other.Mostly,the 15samples were divided into CK and treatment groups,except for the CK-3.The MISP cluster analysis showed the division of CK-1,CK-2and HN-1individuals from the others.Samples of each patch could not be clustered into groups independently;however,the individuals in NN and LN patches showed a closer

relationship among each other,as did the individuals in MN and HN patches (Fig.5b ).

Linear regression between methylation levels and morphological indices

The relationships among total cytosine-methylation levels of the three DNA samples,the changed methylation levels of CG and CHG in each patch and the corresponding morphological indices were investigated.Interestingly,bud number,rhizome length,aboveground biomass,underground biomass and total

CK NN LN MN HN CK NN LN MN HN Hpa II

Msp

I Fig. 3.Example of methylation-sensitive ampli ?cation polymorphism (MSAP)pro ?le,showing the alterations in cytosine methylation at the 50-CCGG sites in the leaf-tissue of Hierochloe grabla ,following multi-gradient nitrogen https://www.360docs.net/doc/c337409.html,nes 1–3represent replicates from the same treatment or control group.The arrows refer to alterations of DNA methylation.The primer combination is ‘3B ’(Table 1).Heterogeneous habitats are indicated by NN,LN,MN and HN,with nitrate added at concentrations of 0,0.15,0.30and 0.45g N kg –1soil,respectively;homogeneous habitats are indicated by CK (control).

3.5

3.02.52.01.51.00.50.0

38.6

38.839.039.239.439.639.840.0(a )

(b )

CK NN LN MN HN Total CG –CG +CHG –CHG +

M e t h y l a t i o n p o l y m o r p h i s m (%)

T o t a l m e t h y l a t i o n c o n t e n t (%)

Fig.4.Methylation-sensitive ampli ?cation polymorphism (MSAP)-based (a )total cytosine methylation content and (b )the methylation polymorphism of the two major types,CG and CHG,of the 50-CCGG sites in the leaf-tissue of Hierochloe glabra ,which were located in nitrogen-addition patches (NN,LN,MN and HN,with nitrate added at concentrations of 0,0.15,0.30and 0.45g N kg –1soil,respectively)and in homogeneous habitat (CK).CG –and CHG –represent hypomethylation;CG+and CHG+represent hypermethylation.Data are the means ?s.e.m.Columns with different letters differ signi ?cantly (P <0.05).

278Australian Journal of Botany R.Bian et al.

biomass were positively correlated with total methylation levels,but negatively with CG hypomethylation.CHG hypomethylation also exhibited signi ?cantly negative correlations with the rhizome length,aboveground biomass and total biomass (Table 2).Other phenotypic traits not present in the table,such as ramet number,ramet height and spacer length,had no substantial relationships with the methylation levels.

Sequence analysis of the differentially methylated DNA bands

The sequences of 27differentially methylated DNA bands were subject to BlastN and BlastX analyses on the NCBI website.As shown in Table 3,the range of fragment sizes was 256–363bp,with an average of 302bp.Among these 27DNA sequences,?ve were homologous to known-function genes encoding NADH dehydrogenase,sterol demethylase,and a hypothetical protein,and one was homologous to an

unknown-function gene.Two sequences were homologous to a putative retrotransposon protein and zinc-knuckle protein revealed by BlastX.Discussion

Genetic differentiation and the consequential phenotypic plasticity were classically considered to be the main strategies for plants to adapt to heterogeneous environments (Grativol et al .2012).More recently,it was found that DNA methylation has the potential to affect plant phenotypes under environmental stimuli (Sherman and Talbert 2002;Aina et al .2004;Verhoeven et al .2010b )and,ultimately,cause ?tness differences among individuals.Therefore,we selected ramets of H.glabra with similar genetic bases to avoid the effect of genetic variation.Phenotypic plasticity is an important strategy for plants to adapt to spatial and temporal environmental heterogeneity (Pigliucci 2005).In the present study,a whole growing season was used to detect whether multi-gradient N supply affects

0.9360.9450.9530.9620.970

CK-1CK-2CK-3NN-1LN-1LN-2NN-2LN-3NN-3HN-2HN-3HN-1MN-1MN-2MN-3

(a )0.9780.9820.986Coefficient

0.9900.994

CK-1CK-2CK-3NN-1LN-1LN-2NN-2LN-3MN-1HN-3NN-3HN-2MN-2MN-3HN-1

(b )

Fig.5.Dendrograms of the leaf-tissue DNA samples derived from Hierochloe glabra located in nitrogen-addition patches (NN,LN,MN and HN,with nitrate added at concentrations of 0,0.15,0.30and 0.45g N kg –1soil,respectively)and the homogeneous habitat (CK).Numbers 1–3represent the replicates from the same treatment or control group.The dendrograms were constructed by the the unweighted pair-group method using arithmetic averages (UPGMA),on the basis of the similarity matrix of (a )methylation-sensitive polymorphism (MSP)and (b )methylation-insensitive polymorphism (MISP)data calculated according to the Jaccard index.

Habitat adaptation through methylation variations Australian Journal of Botany 279

phenotypic variation in the clonal plant,H.glabra.However, only SLA in the MN patch widely differed from that in other patches.SLA,as an indicator of relative growth rate,stress tolerance and photosynthesis,has the potential to re?ect the adaptive strategies of plants in different habitats(Scheepens et al.2010).Clonal plants have the ability to translocate resources among interconnected ramets through physiological integration under heterogeneous habitats(Alpert and Mooney 1986;Hutchings and Wijesinghe1997).Thus,we inferred that more photosynthates produced by ramets in the MN patch with the higher SLA values might be transported among patches in the heterogeneous habitats,which then affect the resource level and morphological traits of those ramets.The plastic morphological responses to environmental heterogeneity can be rapid and sensitive over short-term temporal scales(Roiloa and Retuerto 2006).As shown by the sequential detection of new H.glabra ramets under patchy nutrient environments,pre-existing signi?cant differences in ramet number disappear at the?nal growing stage(Gao et al.2012).In our study,resource heterogeneity might decrease or even disappear over time as N is translocated by rhizomes through the maternal plant.Therefore, the nutrient gradient is not the only factor affecting phenotypic plasticity of clonal plants.

Previous studies have con?rmed that environmental factors such as coldness,heavy metals,aluminium toxicity and salinity tend to cause demethylation of genomic DNA(Steward et al. 2002;Choi and Sano2007;Wang et al.2011).We found that N supply could reduce DNA cytosine-methylation levels of H.glabra,especially for CHG.Cluster analyses further showed that the different amounts of N added not only induced epigenetic divergence between treatment and control plants but also among individuals within treatments.Empirical studies have shown that CHG methylation may relate to defence against transposons and RNA viruses(Hobolth et al.2006).The analyses of differentially methylated DNA bands showed that three sequences were homologous to known-function genes encoding sterol demethylase,which leads to the reduction of methylation levels.The Blast results also showed a homologous putative retrotransposon protein,which represented the most abundant class of transposable elements(TEs).TEs were inactive during normal growth and development but could be activated by demethylation(Hilbricht et al.2008;Johannes et al.2009).Therefore,the higher level of CHG methylation detected in the ramets of homogeneous habitats may effectively suppress the activity of transposons and defend their genome from the deleterious effects of endogenous transposons(Kakutani 2002).

Morphological traits measured in our study were inhibited by the decrease in methylation.This is in accordance with previous studies stating that hypomethylation can cause a signi?cant reduction in ecologically important plant traits (Burn et al.1993;Finnegan et al.1996;Bossdorf et al.2010). For example,the hypomethylation of mangrove individuals (Laguncularia racemosa)near salt marshes resulted in a more signi?cant reduction of morphological traits,such as tree height, tree diameter,leaf width and leaf area,than did that of individuals located near rivers(Lira-Medeiros et al.2010).Mirouze and Paszkowski(2011)suggested that alterations of epigenetic regulation in plant populations with limited genetic diversity could lead to the formation of heritable epialleles,transcription and mobilisation of transposable elements.Epialleles and transposon-driven variation in gene expression generated

Table2.Linear regression relationships between methylation levels

and morphological indices of Hierochloe glabra under multi-gradient

nitrogen supply

BN,bud number(#);RL,rhizome length(cm);AB,aboveground biomass(g);

UB,underground biomass(g);TB,total biomass(g).*P0.05,**P0.005

Independent variable Regression equation R2

Total methylation level

BN y=933.470x–352.0230.905*

RL y=18903.513x–7151.7000.958**

AB y=101.122x–38.7510.932**

UB y=199.163x–75.7090.934**

TB y=300.285x–114.4600.963**

CG-hypomethylation level

BN y=–1144.271x+26.8010.821*

RL y=–23980.764x+527.6720.930**

AB y=–130.023x+2.3460.929**

UB y=–247.944x+5.1530.874*

TB y=–377.967x+7.4990.920*

CHG-hypomethylation level

RL y=–48947.106x+451.8000.772*

AB y=–273.153x+1.9600.817*

TB y=–794.581x+6.3780.810*

Table3.Sequence analysis of differentially methylated fragments of Hierochloe glabra,based on BlastN and BlastX on the NCBI website

MSAP,methylation-sensitive ampli?cation polymorphism

MSAP bands Size(bp)Function based on BlastX against the

NCBI database GenBank,ID E-value

Identity based on BlastN against the NCBI

database GenBank,ID E-value

1274No signi?cant similarity found Bambusa oldhamii NADH dehydrogenase,EU365401.11e-113

2305Putative retrotransposon protein

(Phyllostachys edulis),ADB85398.17e-32

No signi?cant similarity found

3318No signi?cant similarity found Avena strigosa sterol demethylase,DQ680849.17e-81

4258No signi?cant similarity found Oryza sativa Japonica Group hypothetical protein,AP006441.31e-116 5358No signi?cant similarity found Avena strigosa sterol demethylase,DQ680849.12e-82

6256Zinc knuckle protein(Oryza sativa Japonica

Group),AAX95626.16e-05

No signi?cant similarity found

7299No signi?cant similarity found Avena strigosa sterol demethylase,DQ680849.17e-81

8304No signi?cant similarity found Secale cereale clone R1–5genomic sequence,DQ414510.10.001 280Australian Journal of Botany R.Bian et al.

phenotypic diversity for natural selection(Mirouze and Paszkowski2011).Therefore,the decreased DNA methylation might be considered the main cause of the phenotypic reduction in bud number,rhizome length and biomass(Table2).Moreover, bud number and rhizome length were important indices that affected the vegetative propagation and expansion ability of clonal plants(Wang et al.2008;Gao et al.2012).In other words,higher methylation levels of ramets in the CK patch might activate the potential for asexual reproduction to produce more offspring and expand the population.This adaptive strategy may explain the appearance of H.glabra as a main species in the meadow steppe of Songnen Plain,where severe patchy resource distribution occurs.

There is increasing evidence that phenotypic variation can be caused by variations in epigenetic modi?cations of the genome(Johannes et al.2009;Mar?l et al.2009;Bossdorf et al.2010;Zhang et al.2013).However,most of these studies have focussed on model plants under controlled conditions.More?eld studies on non-model species are required to determine the effects under natural conditions. This will be particularly interesting and will stress the ecological and evolutionary importance of epigenetics in real-world contexts.

Acknowledgements

This study was supported?nancially by the National Natural Science Foundation of China(No.31070375)and the Natural Scienti?c Foundation of Jilin Province,China(No.20101556;20100150).The authors thank engineer Baotian Zhang for assistance in our greenhouse experiment and Dr Linfeng Li for valuable comments on earlier drafts. References

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