Reserch Intrspecific genetic diversity modultes plnt soil feedck nd nutrient cycling Mrin Semchenko 1,2, Sirgi Sr 1,2 nd Anu Lepik 2 1 School of Erth nd Environmentl Sciences, The University of Mnchester, Oxford Rod, Mnchester, M13 9PT, UK; 2 Deprtment of Botny, Institute of Ecology nd Erth Sciences, University of Trtu, Li 4, Trtu 515, Estoni Author for correspondence: Mrin Semchenko Tel: +44 11612751152 Emil: mrin.semchenko@mnchester.c.uk Received: 21 Mrch 217 Accepted: 7 My 217 doi: 1.1111/nph.14653 Key words: decomposition, ecosystem functioning, functionl trits, genetic diversity, intrspecific vrition, kin recognition, phenotypic plsticity, soil feedck. Summry Plnt genetic diversity cn ffect ecosystem functioning y enhncing productivity, litter decomposition nd resistnce to nturl enemies. However, the mechnisms underlying these effects remin poorly understood. We hypothesized tht genetic diversity my influence ecosystem processes y eliciting functionl plsticity mong individuls encountering kin or geneticlly diverse neighourhoods. We used soil conditioned y groups of closely relted (silings) nd diverse genotypes of Deschmpsi cespitos species known to exhiit kin recognition vi root exudtion to investigte the consequences of kin interctions for root litter decomposition nd negtive feedck etween plnts nd soil iot. Geneticlly diverse groups produced root litter tht hd higher nitrogen (N) content, decomposed fster nd resulted in greter N uptke y the next genertion of seedlings compred with litter produced y siling groups. However, similr degree of negtive soil feedck on plnt productivity ws oserved in soil conditioned y silings nd geneticlly diverse groups. This suggests tht chrcteristics of roots produced y siling groups slow down N cycling ut moderte the expected negtive impct of soil pthogens in low-diversity stnds. These findings highlight interctions etween neighouring genotypes s n overlooked mechnism y which genetic diversity cn ffect iotic soil feedck nd nutrient cycling. Introduction Plnt species diversity nd ssocited vrition in functionl trits re centrl to the mintennce of ecosystem services, such s primry production, cron storge nd nutrient cycling (Knops et l., 22; Steineiss et l., 28; Cong et l., 214). Speciesrich plnt communities lso experience reduced pthogen lod nd specilist herivore dmge compred with species monocultures (Mitchell et l., 22; Lu et l., 28; Schnitzer et l., 211). Although the importnce of species diversity is well estlished, there is incresing evidence tht intrspecific genetic diversity plys n importnt role in mintining species co-existence (Booth & Grime, 23; Whitlock, 214; Schoe et l., 215) nd enhncing net primry productivity (Crutsinger et l., 26), litter decomposition (Schweitzer et l., 25), nd resistnce to dmge y herivores nd folir pthogens (Zhu et l., 2; Prker et l., 21; Tooker & Frnk, 212; Brton et l., 215). The mechnisms linking intrspecific genetic diversity with ecosystem functioning re still poorly understood, not lest ecuse geneticlly diverse popultions my enhnce or inhiit ecosystem processes to greter degree thn predicted sed on the effects of constituent genotypes in isoltion (Hughes et l., 28). Nondditive effects of genetic diversity on ecologicl processes my e explined y numer of mechnisms. Primry productivity might e enhnced y resource niche prtitioning mong diverse plnt genotypes, lthough empiricl evidence for this is lcking (Crutsinger et l., 26; Atwter & Cllwy, 215). The effect of plnt genetic diversity on ecosystem processes my lso e medited y microil nd inverterte composition nd ctivity in the soil. Mixed-genotype plnt litter hs een shown to decompose fster thn expected from the decomposition rtes of single-genotype litters (Schweitzer et l., 25; Wng et l., 214). This effect hs een ttriuted to resource complementrity wherey mixed litter provides more complete rnge of sustrtes to more diverse set of decomposers. How the incresed rtes of decomposition in genotypic mixtures ffect the lnce etween nitrogen minerliztion nd immoiliztion is not known ut significnt feedck effects on plnt nutrition could e expected. In ddition to effects medited y nutrient cycling, genotypic diversity within species could reduce negtive plnt soil feedck y slowing down the ccumultion of soil pthogens, s hs een shown for diversity t the species level (Mron et l., 211; Schnitzer et l., 211). This prediction is supported y studies tht demonstrte genotypic nd popultion specificity of plnt 9 Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust
New Phytologist Reserch 91 interctions with ntgonistic soil iot (Liu et l., 215; Luo et l., 216), nd reduced levels of oveground herivory nd lef pthogen dmge in genotypic mixtures compred with monocultures (Prker et l., 21; Tooker & Frnk, 212; Brton et l., 215). The ltter finding hs een ttriuted to the difficulty for nturl enemies in spreding to susceptile genotypes nd/or induced defence in susceptile genotypes when growing mong resistnt genotypes (ssocitionl resistnce; Bros et l., 29). In ddition, plnt genotypes my express different phenotypes when grown in mixtures compred with genotypic monocultures, nd such plsticity my hve consequences for ecosystem processes. In prticulr, severl studies hve demonstrted the ility of plnts to recognize kin vi root exudtes nd voltiles (Biedrzycki et l., 21; Krn et l., 213; Semchenko et l., 214) nd modify their growth, iomss lloction nd morphology in response to the genetic reltedness of neighours (Dudley & File, 27; Bhtt et l., 21; Biernskie, 211; Lepik et l., 212). Such plsticity represents specil cse of indirect genetic effects, which re defined s the effect of genotype on the phenotype of neighouring conspecifics (Mutic & Wolf, 27; Cost e Silv et l., 213). Interctions with kin re likely to increse selection for resource-conservtive strtegy wherey resources re diverted wy from wsteful competition with closely relted individuls towrds investment into tissue longevity nd defence ginst nturl enemies, which re known to spred fster in geneticlly homogenous compred with diverse stnds (File et l., 212,; Semchenko et l., 213). Improved tissue longevity nd protection cn e chieved y the production of thicker leves nd roots with higher tissue density nd lower concentrtion of nitrogen reltive to cron compounds, such s lignin (Tjoelker et l., 25; Roumet et l., 216). Such phenotypic chnges cn hve cscding effects on soil microil ctivity nd composition, which cn, in turn, feed ck to influence plnt phenotype, nutrition nd fitness (Whithm et l., 26; Mdritch & Lindroth, 211; Bry et l., 212). Thus, plnt responses to the genetic reltedness of neighours represent process with strong potentil to influence ecosystem functioning. However, its role hs not een explicitly tested. In the present study, we test the hypothesis tht plnt responses to the genetic reltedness of surrounding individuls nd ssocited chnges in functionl trits cn hve significnt consequences for nutrient cycling nd feedck etween plnts nd soil iot. The study uilds on our previous discovery tht root exudtes of geneticlly diverse individuls of Deschmpsi cespitos trigger the production of longer, more rnched roots with higher specific root length (SRL), wheres siling exudtes suppress such response (Semchenko et l., 214). As low SRL is ssocited with slow growth nd high tissue longevity, this finding likely reflects switch to more resource-conservtive strtegy: low SRL in the presence of closely relted neighours rises construction costs of resource-cpturing orgns ut my reduce susceptiility to pthogen ttck nd decomposition (Tjoelker et l., 25; Reich, 214; Lemmermeyer et l., 215; Roumet et l., 216). We therefore predicted tht (1) plnts grown in groups of closely relted individuls produce low qulity root litter tht decomposes more slowly compred with groups of geneticlly diverse plnts; nd (2) chnges in root properties in response to interctions with kin mitigte the stronger negtive plnt soil feedck expected in stnds with low genetic diversity. This ws tested y growing D. cespitos plnts in either groups of silings or geneticlly diverse individuls nd following the decomposition of produced root litter, nd the growth of nd N uptke y the next genertion of seedlings in soils conditioned y plnts of different identities. Mterils nd Methods Experimentl design Soil conditioning phse Seeds from multiple mother plnts of Deschmpsi cespitos (L.) P. Beuv. were collected from flooded medow in Estoni with totl re of c. 4 h (58 25 32 N; 26 3 4 E). Plnts were grown s either siling groups (offspring of the sme mother plnt) or geneticlly diverse groups composed of individuls from multiple mother plnts from the sme popultion (Fig. 1). In the siling tretment, 1 pots were sown with seeds from 1 seed heds ( single seed hed per pot), ech collected from different mother plnt seprted from ll others y t lest 2 m. Pots with geneticlly diverse plnt groups were replicted six times nd were ssemled using seed mixture from the sme ten loctions s those used in the siling tretment Conditioning phse Feedck phse Silings Diverse Control +/ steriliztion +/ steriliztion Soil nlysis Root C & N Soil feedck: Seedling growth Totl mss C & N Root decomposition Fig. 1 Experimentl design. In the conditioning stge, plnts of Deschmpsi cespitos were grown in groups of either silings or mixture of genotypes originting from different mother plnts from the sme popultion. In the control tretment, pots were filled with the soil mixture ut no seedlings were plnted. After 3 months, hlf of the conditioned soil ws sterilized y gmm rdition. Soil nd root smples were collected for nlysis. In the feedck stge, new seedlings were plnted into control soil or soil conditioned y either silings or geneticlly diverse groups. Seedling growth t 2 wk nd totl dry mss nd tissue nitrogen (N) nd cron (C) content fter 8 wk of growth were mesured to ssess plnt responses to soil conditioning nd steriliztion. Conditioned soil included ded roots, llowing estimtion of root litter decomposition during the feedck stge of the experiment. Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust
92 Reserch New Phytologist 1 seed heds used in the siling tretment long with dditionl seed heds from the immedite vicinity (< c. 2 m). Additionl seed heds were used in the geneticlly diverse groups ecuse the ten seed heds used to generte the siling groups did not provide sufficient seeds for oth conditioning nd feedck stges of the experiment. Deschmpsi cespitos is selfincomptile grss species tht forms dense tussocks of geneticlly identicl shoots ut does not exhiit long-distnce clonl dispersl (Hlevy, 2). It is therefore highly likely tht seeds collected from shoots growing > 2 m prt represent different genotypes. Seeds of pproprite identity were sown on the surfce of 5-l pots filled with 1 : 1 mixture of fine snd nd commercilly ville soil (ph 6.5, wter-solule N 1 mg l 1, P 8 mg l 1, K 4 mg l 1 ). Nturl soil from the home site ws dded t the rte of 25 g l 1 of soil mixture to provide plnts with nturl soil iot. Three weeks fter seed sowing, seedlings were thinned to leve 15 plnts in ech pot. In ddition, six pots were filled with the sme soil mixture ut no seedlings were plnted in these pots to crete control tretment. All pots were plced rndomly on ench in glsshouse with 16 h : 8 h, dy : night illumintion cycle. All pots were hrvested fter 3 months. Shoots were cut t soil level nd removed ut roots remined in the conditioned soil. Soil from ech pot ws roken up, mixed nd split into two equl prts, nd one prt ws sterilized using gmm rdition (dose 15 kgy). Soil from ech siling group ws kept seprtely to mintin genetic reltedness etween plnts in the conditioning nd feedck stges, wheres control soil nd soil conditioned y geneticlly diverse groups were ech pooled within ech steriliztion tretment nd split etween 1 replicte pots for the feedck experiment. Further detils on soil hndling re presented in Supporting Informtion Methods S1. Three soil smples were tken t rndom from ech conditioning (silings, geneticlly diverse, control) nd steriliztion (sterile nd nonsterile) tretment. Within the siling tretment, the sme three rndomly selected siling groups were smpled from sterile nd nonsterile tretments. Roots were seprted from the soil, dried, nd root cron (C) nd nitrogen (N) content ws determined using CN elementl nlyser (Elementr vrio EL cue; Elementr Anlysensysteme GmH, Lngenselold, Germny). Soil ph KCl nd N (Kjeldhl method), ville phosphorus (P), ville potssium (K) nd orgnic mtter content were determined ccording to methods descried in Moore & Chpmn (1986). Soil feedck experiment Seeds collected from the sme 1 mother plnts s those used to crete siling groups in the conditioning phse were germinted on moist snd. Seven dys lter, six seedlings from ech mother plnt were trnsplnted ( single seedling per pot) into the centre of 1.3-l pots filled with soil (.57 kg DW) tht ws (1) conditioned y silings, geneticlly diverse groups or no plnts (control), nd (2) sterilized or left unsterilized (Fig. 1). Soil conditioned y siling groups ws occupied in the feedck stge y seedling from the sme mternl linege. This resulted in fctoril design of three conditioning nd two steriliztion tretments, with 1 replictes for ech tretment comintion (6 pots in totl). All plnts were plced rndomly on enches in glsshouse with 16 h : 8 h, dy : night illumintion cycle. The position of pots ws re-rndomized every 2 wk. As the soil hd een extensively exploited y dense vegettion during the conditioning phse, plnts were fertilized four times during the feedck stge with 2 ml of.7% solution of liquid fertiliser (6% N, 2.18% P, 4.15% K) in the feedck stge. This prtilly replenished nutrients tht were removed from the soil during the conditioning stge. In the nturl hitt of the study species, nutrient deposition occurs in the form of yerly flooding. Two weeks fter trnsplnttion, the length of the longest lef ws registered for ech seedling to exmine the effect of soil feedck t erly stges of seedling estlishment. Plnts were hrvested fter 58 d of growth. Roots were crefully wshed out of the soil nd seprted into roots elonging to focl plnts (ttched to oveground shoots nd different in colour nd texture from ded roots) nd ded roots tht were left undecomposed in the conditioned soil. In our experiment, we followed the pproch for in situ decomposition estimtion s proposed y Dornush et l. (22). Nmely, the ded roots from the conditioning stge were left to decompose in the soil undistured, rther thn eing wshed out, dried, weighed nd plced in litter g. Although not s precise s estimtes otined using litter gs, this pproch retins relistic conditions of root litter density nd sptil distriution, nd llows close contct etween ded roots, soil nd the live roots of newly plnted seedlings. Soil from ech pot in the conditioning stge ws divided into two equl prts, nd one prt ws sterilized with gmm rdition. As decomposition ws lrgely suppressed in sterilized soil (lthough some microil coloniztion proly occurred in the course of experiment), comprison of the remining ded root mss in nonsterile nd sterile soil t the end of the feedck stge provided n estimte of root decomposition. Initil root litter mss for ech conditioned soil ws estimted using shoot iomss dt from the conditioning stge nd the men root-to-shoot iomss rtio from previous study tht used the sme soil nd mternl lines s in the current study, s well s similr growth conditions nd durtion (Semchenko et l., 214). Using predicted initil root mss nd litter mss remining in soil t hrvest, we clculted the proportion of litter mss remining fter 58 d of incution. All plnt mteril ws dried t 7 C for 48 h nd weighed seprtely. Root nd shoot C nd N content of focl plnts were determined using n Elementr vrio EL cue nlyser (Elementr Anlysensysteme GmH). An dventitious root rnch with ll ttched higher order rnches ws selected from live roots nd scnned (Epson Perfection V7 Photo; Epson, Suw, Jpn) nd the totl root length ws clculted using WINRHIZO PRO 28 (Regent Instruments Inc., Queec City, QC, Cnd). Specific root length (SRL) ws clculted s the rtio of the root length to the dry mss of the scnned root smple. Trit dt re presented in Tle S1. Sttisticl nlysis Soil conditioning phse Liner fixed effects models were used to ssess the effects of conditioning tretment (silings vs Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust
New Phytologist Reserch 93 geneticlly diverse groups) on root C nd N content nd C : N rtio. Similrly, liner fixed effects models were used to estimte the effects of soil conditioning (silings, geneticlly diverse groups, control) nd steriliztion on soil ph, N, P, K nd orgnic mtter content t the end of the conditioning phse. Soil feedck experiment Liner mixed models were used to estimte the effects of soil conditioning (silings, geneticlly diverse groups) nd steriliztion (sterile, nonsterile) on ech of the following response vriles in the feedck experiment: (1) proportion of root litter remining fter 8 wk of incution, (2) initil seedling growth t 2 wk (mximum lef length), (3) totl dry mss t hrvest, (4) root : shoot iomss rtio, (5) specific root length nd (6) C nd N content nd C : N rtio of focl plnts (root nd shoot). Mother plnt linege ws included in the nlysis s rndom fctor. Dt were log e -trnsformed (or squre root-trnsformed for the proportion of litter mss remining) efore nlysis if necessry to stisfy model ssumptions. Distriutionl ssumptions were checked y exmining norml proility plots, nd vrinces were compred using Levene s test. The effect of the soil steriliztion tretment reflects the result of eliminting soil iot ut lso potentil flush of nutrients from ded microil cells (McNmr et l., 23). To otin informtion on the role of soil iot ssocited with plnts without the confounding effect of nutrient flush, we clculted soil feedck index tht compred plnt trits in soil conditioned y different plnt groups to soil tht ws unoccupied y plnts during the conditioning stge. The soil feedck index ws clculted s ln(t tret /T control ), where T tret is the trit men for plnts in ech soil conditioning 9 steriliztion tretment comintion (soil conditioned y silings or geneticlly diverse groups nd either sterile or nonsterile soil) nd T control is the trit men for plnts grown in the corresponding control soil (no plnts in the conditioning stge nd either sterilized or left unsterilized). Negtive vlues indicte reduced plnt performnce in soil conditioned y plnts compred to control soil tht ws not occupied y ny plnts in the conditioning stge. Mens nd stndrd errors for the soil feedck index were otined s prmeter estimtes from liner mixed models contining the three soil conditioning tretments (fixed fctor: silings, geneticlly diverse groups nd control s the reference level) nd mother plnt linege s rndom fctor. Models were performed seprtely for sterile nd nonsterile soil to llow comprisons to e mde with the pproprite control soil. For seedling length nd root C : N rtio, the index ws clculted on untrnsformed dt (T tret T control ) s log e -trnsformtion resulted in skewed distriution. All nlyses were performed using R v.3.3.2 (R Core Tem, 216). Mixed models were performed using the LME4 pckge (Btes et l., 215). Results Root decomposition nd C/N content The proportion of root litter remining in the soil t the end of the feedck stge ws similr etween plnt diversity tretments when soil ws sterilized. In unsterilized soil, significntly greter proportion of root litter ws lost from soil conditioned y geneticlly diverse groups compred with soil conditioned y siling plnts (79% vs 48% litter mss loss, respectively; significnt interction etween the effects of soil conditioning nd steriliztion in Tle 1; Fig. 2). Proportion of litter remining.75 1.5.25 Sterile Silings Nonsterile Diverse Fig. 2 The effect of soil conditioning nd steriliztion on the proportion of Deschmpsi cespitos root litter mss remining in soil fter 8 wk of incution. Soil ws conditioned y either silings or mixture of genotypes originting from different mother plnts from the sme popultion, nd ws either left unsterilized (closed rs) or sterilized y gmm rdition efore incution (open rs). Mens SE re shown. Sttisticl nlysis ws performed on squre-root trnsformed dt. Different letters indicte significnt differences etween mens (P <.5, Tukey s test). Tle 1 The effect of soil conditioning (silings or geneticlly diverse groups) nd soil steriliztion on Deschmpsi cespitos seedling growth t 2 wk, totl plnt dry mss nd nitrogen (N), root N% nd the proportion of litter mss remining in soil fter 8 wk of incution df Prop. litter remining Root N% Totl N (g) Seedling length (cm) Plnt mss (g) Conditioning (C) 1 4.9* 8.4** 1.2.1.6 Steriliztion (S) 1 33.8*** 54.*** 49.5*** 25.8*** 11.6*** C 9 S 1 6.5* 8.9** 13.9*** 2..1 Residuls 27 F-vlues nd their significnce re shown: *, P <.5; **, P <.1; ***, P <.1. Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust
94 Reserch New Phytologist Groups of silings nd geneticlly diverse plnts produced similr totl oveground iomss y the end of the conditioning stge (men mss 25.3 g nd 24.7 g, respectively; P =.7329, t-test). The roots of plnts grown in groups of geneticlly diverse individuls in the conditioning stge hd 28% higher N content thn roots produced when grown mong silings, resulting in considerly lower root C : N rtio in geneticlly diverse groups compred with siling plnts (Tle 2). These differences in root N content crried over to the next genertion of plnts in the feedck stge, ut were only detectle in the unsterilized soil tretment (interction etween the effects of soil conditioning nd steriliztion, N% F 1,27 = 8.9, P =.6; C : N F 1,27 = 8., P =.88; C% F 1,27 = 2.4, P =.1353; totl plnt N F 1,27 = 13.9, P =.9; Tle 1). Plnts grown in unsterilized soil conditioned y groups of geneticlly diverse individuls produced roots with significntly higher N content nd significntly lower C : N rtio compred with plnts grown in soil conditioned y silings (oth differences P <.5, Tukey test; Figs 3, S1). The min cuse for differences in C : N rtios ws Tle 2 Root cron (C) nd nitrogen (N) content of Deschmpsi cespitos plnts grown in either groups of silings or geneticlly diverse individuls from the sme popultion Tretment Silings Diverse C% F 1,1 = 3.8 $ 45.45 (.14) 44.77 (.32) N% F 1,1 = 6.*.57 (.2).74 (.6) C:N F 1,1 = 8.7* 8.31 (3.31) 62.76 (4.94) F-vlues, the significnce of the tretment ( $, P <.1; *, P <.5), rw mens nd stndrd errors (in prentheses) re shown. the higher root N content of plnts grown in soil conditioned y geneticlly diverse groups nd slight increse in C content in the roots of plnts grown in soil conditioned y silings reltive to those grown in the unconditioned control soil (i.e. soil tht ws not occupied y ny plnts in the conditioning stge; oth differences P <.5, t-test; Figs 3e, S1). Totl N uptke of plnts (shoots nd roots comined) grown in unsterilized soil conditioned y geneticlly diverse groups ws similr to uptke y plnts grown in unconditioned control soil, wheres plnts in soil conditioned y silings exhiited significntly lower N uptke compred with the unconditioned control nd geneticlly diverse tretments (P <.5; Fig. 3,f). Soil feedck effects on plnt growth Initil seedling growth ws not significntly ffected y soil conditioning (Tle 1; Fig 3c). A mrginlly nonsignificnt suppression of growth in soil conditioned y silings compred to unconditioned control ws oserved in the unsterilized tretment (Fig. 3g). After 8 wk of growth, plnt growth ws significntly improved y soil steriliztion (Tle 1; Fig. 3d). Compred with plnt iomss in soil tht ws not occupied y plnts in the conditioning stge, plnt growth ws strongly suppressed in unsterilized soil nd not significntly ffected in sterile soil (Fig. 3h). This suggests tht plnt presence in the conditioning stge modified soil iot with negtive impct on plnt growth in the feedck stge of the experiment. The strength of growth suppression ws similr in soils conditioned y silings nd geneticlly diverse groups. () Sterile ().8 (c) 1 1.5 Nonsterile c c (d) 8.6 c 1 6.4 4.5.2 2 Root N% Totl N (g) Seedling length (cm) Plnt dry mss (g) 6 4 2 (e).5 (f).2 (g) 1 (h) Root N feedck.4.3.2.1 *** Totl N feedck Seedling feedck.2.2 $ 1.4.4 * 2 $ ** $.6.6 * Silings Diverse Silings Diverse Silings Diverse Silings Diverse Fig. 3 The effect of soil conditioning nd steriliztion on () root nitrogen (N) content, () totl plnt N, (c) seedling length fter 2 wk of growth, nd (d) plnt dry mss fter 8 wk of growth; nd soil feedck for (e) root N content, (f) totl N, (g) seedling length nd (h) plnt mss of Deschmpsi cespitos in comprison to n unconditioned control tretment. Plnts were sujected to soil tht ws conditioned y either silings from the sme mother plnt or mixture of genotypes originting from different mother plnts from the sme popultion. Conditioned soil ws either left unsterilized (closed rs) or sterilized y gmm rdition efore plnting the next genertion of seedlings (open rs). For plnt mss nd N dt, sttisticl nlysis ws performed on log e -trnsformed dt. Different letters indicte significnt differences etween mens (P <.5, Tukey s test). Soil feedck ws clculted s log e - trnsformed rtio of men root N (e), totl N (f) or plnt dry mss (g) or s the difference in men seedling length (h) etween conditioned soil nd unconditioned control. Mens SE re shown. Symols indicte significnt differences from the control: $, P <.1; *, P <.5; **, P <.1; ***, P <.1 (t-test). Dry mss feedck Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust
New Phytologist Reserch 95 Effects of soil conditioning on iomss lloction nd root morphology The root : shoot iomss rtios of plnts in the feedck stge of the experiment were not significntly ffected y soil steriliztion nd plnt diversity in the conditioning stge (ll effects P >.1; Tle 3). There ws mrginlly nonsignificnt interctive effect of soil steriliztion nd plnt diversity tretments on SRL (F 1,27 = 3.9, P =.598; Tle 3). When soil ws sterilized, plnts produced roots with lower SRL when grown in soil conditioned y silings compred with soil conditioned y diverse plnt groups. In unsterilized soil, SRL ws similr in soil conditioned y silings nd geneticlly diverse groups. Effects of soil conditioning nd steriliztion on soil properties Soil conditioned y groups of geneticlly diverse individuls nd silings oth resulted in significntly lower soil orgnic mtter content nd higher ph compred with soil tht remined unoccupied y plnts during the conditioning stge (significnt effect of soil conditioning F 2,14 = 11.7, P =.1 nd F 2,14 = 128.9, P <.1, respectively; Tles S2, S3). Soil conditioned y silings exhiited higher K content thn soil conditioned y geneticlly diverse groups (significnt effect of soil conditioning F 2,14 = 16.7, P =.2) ut did not differ significntly in N nd P vilility, orgnic mtter content, or ph (P >.5; Tles S2, S3). Discussion In the present study, we tested whether phenotypic plsticity to neighour identity cn medite the effects of plnt genetic diversity on ecosystem processes y triggering chnges in root chemicl properties, with consequences for nutrient cycling nd plnt soil interctions. We found tht plnts produced roots with higher nitrogen (N) content when grown mong geneticlly diverse individuls compred with groups of closely relted silings. Also, root litter produced in geneticlly diverse groups decomposed fster nd resulted in greter N uptke y the next genertion of seedlings compred with litter produced in siling groups. However, plnt iomss ccumultion ws eqully suppressed in soils conditioned y geneticlly diverse nd siling groups compred with soils tht were unoccupied y ny plnts in the conditioning Tle 3 The effect of soil steriliztion nd soil conditioning (silings or geneticlly diverse groups) on specific root length (SRL) nd lloction of iomss etween roots nd shoots in the next genertion of Deschmpsi cespitos seedlings Root : shoot rtio SRL (cm mg 1 ) Nonsterile Silings.45 (.3) 47.5 (4.8) Diverse.34 (.2) 44. (5.3) Sterile Silings.41 (.3) 32.6 (5.1) Diverse.42 (.5) 47. (5.6) Rw mens nd stndrd errors (in prentheses) re shown. Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust stge. This suggests tht the slower N cycling oserved in lowcompred with high-diversity stnds my hve een offset y superior ility of plnts to resist negtive feedck from soil iot, which is reported frequently in soils conditioned y conspecifics (Mron et l., 211; Schnitzer et l., 211). Consequences for nutrient cycling Severl studies hve now demonstrted tht plnts respond to the genetic composition of their neighourhoods, including genetic reltedness within species (Murphy & Dudley, 29; Biedrzycki et l., 21; Lepik et l., 212) nd neighour species identity (Mhll & Cllwy, 1992; Semchenko et l., 27; Akumov et l., 216). If these responses led to chnges in lef or root litter qulity, modified rtes of decomposition nd nutrient cycling cn e expected (Genung et l., 213). However, experiments investigting the effects of genetic diversity on litter decomposition nd nutrient cycling hve lmost exclusively used rtificilly mixed litter from genotypes tht were grown in isoltion (e.g. Mdritch & Hunter, 25; Schweitzer et l., 25; Mdritch et l., 26; Crutsinger et l., 29). Our results show tht such n pproch overlooks the role of iotic interctions nd indirect genetic effects in determining litter qulity nd mediting the reltionship etween genetic diversity nd ecosystem functioning. We found tht root litter produced in groups of geneticlly diverse individuls ws chrcterized y higher N content nd lower cron (C) : N rtio compred with litter produced in groups of silings, which trnslted into fster rtes of litter decomposition nd higher N uptke y seedlings in the next genertion. Notly, differences in N uptke were evident only in unsterilized soil. This suggests tht dditionl N in seedlings grown in soil conditioned y geneticlly diverse plnts ws derived from decomposing root litter. Deschmpi cespitos is dominnt species in its home community nd frequently intercts with conspecifics (Lepik et l., 212; Semchenko et l., 213). The pronounced effects of genotypic diversity in this species my thus e expected to hve importnt consequences throughout the ecosystem. It is likely tht plnt genetic diversity left positive legcy effect on nutrient cycling not only vi higher litter qulity, ut lso y supporting more diverse decomposer community. Plnt genotypes cn support distinct soil microil communities due to differences in the chemicl composition of root exudtes nd litter (Micllef et l., 29; Mdritch & Lindroth, 211), which my e further modified y interctions with multiple genotypes occupying the sme soil spce. Further mnipultive experiments re needed to scertin the reltive contriution of litter properties nd decomposer communities s meditors of plnt diversity effects on nutrient cycling. To dte, the vst mjority of studies hve reported kin recognition sed on the oservtion tht plnts modified their phenotype in response to the genetic reltedness of neighours (reviewed in File et l., 212; Lepik et l., 212). However, it is often uncler if the response to kin ws result of neighour perception vi specific cues or ws medited y resource competition. According to niche prtitioning theory, closely relted plnts re expected to hve more similr resource niches nd
96 Reserch New Phytologist experience stronger competition compred to geneticlly diverse groups (McArthur & Levins, 1967; Silvertown, 24). Greter niche overlp nd competition for limiting nutrients mong silings should result in lower popultion-level productivity nd greter lloction to fine root production. By contrst, we found no differences in iomss production in this study nd hve recorded slightly greter iomss ccumultion nd decresed fine root production in siling groups in erlier studies (Lepik et l., 212; Semchenko et l., 214). We lso showed for the sme popultion of D. cespitos tht fine root production ws suppressed even in the sence of direct competition for resources when exposed to the root exudtes of silings compred with nonsilings (Semchenko et l., 214). However, it is possile tht resource uptke dynmics nd interctions with soil orgnisms (File et l., 212; Pickles et l., 217) interct with responses to neighour identity nd jointly contriute to the effects of genetic diversity on ecosystem functioning. Consequences for iotic soil feedck It hs een shown previously tht higher genetic diversity of plnt popultions results in lower nturl enemy spred nd dmge oveground (ssocitionl resistnce; Zhu et l., 2; Tooker & Frnk, 212; Brton et l., 215). It could therefore e expected tht soil pthogens lso ccumulte fster nd cuse greter dmge when plnts re grown in closely relted compred with geneticlly diverse groups (Luo et l., 216). Greter negtive impct of soil iot on plnt growth in stnds with low genetic diversity my lso e due to stronger competition with soil microes for nutrients. We found tht the growth of D. cespitos seedlings ws eqully suppressed in soils conditioned y silings nd geneticlly diverse individuls reltive to control soil tht remined unvegetted during the conditioning stge. When soils were sterilized y gmm rdition, plnt growth ws no longer suppressed, suggesting tht the oserved negtive soil feedck ws cused y plnt-ssocited soil iot. The sence of differences in plnt iomss ccumultion etween soils conditioned y silings nd geneticlly diverse groups is intriguing given tht the ltter soil enled higher plnt N uptke. Therefore, despite the positive effect of litter decomposers on plnt nutrition, other soil orgnisms seemingly prevented plnts from chieving greter iomss in soil previously occupied y geneticlly diverse plnts compred with soil conditioned y silings. In previous study, we found evidence for kin recognition in the study species, D. cespitos, which triggered production of roots with lower specific length when sujected to siling root exudtes (Semchenko et l., 214). The current study shows tht plnts grown together with silings lso produce roots with higher C : N rtio. This chnge in root properties reflects shift from mximizing growth rtes towrds greter longevity (Tjoelker et l., 25; Reich, 214) nd my mke roots less susceptile (nd ttrctive) to soil pthogens (Herms & Mttson, 1992; Lemmermeyer et l., 215). Therefore, it is possile tht the expected pttern of greter nturl enemy dmge in loctions with low genetic diversity my e prtilly offset in species cple of recognizing kin nd shifting ville resources from competition to other functions, such s defence nd longevity. However, detiled nlysis of root chemicl composition nd undnce of soil pthogens is necessry to evlute this hypothesis. In the present study, soil conditioned y siling groups ws occupied in the feedck stge y seedling from the sme mternl linege to represent scenrio where low genetic diversity t the popultion level nd/or poor seed dispersl result in closely relted individuls occupying the sme soil ptch in consecutive genertions. Besides the perception of their current neighours, plnts lso my e ffected y identity cues left y previous genertions (Mzzoleni et l., 215). We found mrginlly nonsignificnt interctive effect of plnt genetic diversity nd soil steriliztion on the root morphology of seedlings in the next genertion. Plnts hd very similr root morphology in unsterilized soil ut hd lower specific root length when grown in sterilized soil conditioned y silings. A similr response ws oserved in previous study when plnts were exposed to root exudtes produced y silings (Semchenko et l., 214). This finding suggests tht neighour identity cues my hve remined in the soil fter plnt deth ut were modified or wekened y soil microes in unsterilized soil. Conclusions This study proposes novel mechnism y which intrspecific genetic diversity my ffect ecosystem functioning: some plnt species exhiit n ility to shift their resource lloction etween growth- nd longevity-relted functions depending on the genetic diversity of their neighourhoods, with consequences for litter decomposition, nutrient cycling nd plnt soil feedck. This finding highlights the need to ccount for functionl vriility within plnt genotypes when linking diversity to ecosystem processes, nd to work towrds integrting reserch on phenotypic plsticity with community nd ecosystem ecology. Acknowledgements We thnk John Dvison, Richrd Brdgett nd Kristjn Zoel for providing vlule comments on the mnuscript. Jn Aruv li nd Deorh Ashworth provided technicl support with the chemicl nlysis of plnt smples. This study ws supported y the University of Mnchester, Estonin Science foundtion (grnt 9332) nd Institutionl Reserch Funding (IUT 2-31) of the Estonin Ministry of Eduction nd Reserch. Author contriutions M.S. nd A.L. designed nd crried out the experiment; M.S. nd S.S. nlysed the dt; nd M.S. nd S.S. wrote the pper with input from A.L. References Akumov M, Zoel K, Lepik A, Semchenko M. 216. Plsticity in plnt functionl trits is shped y vriility in neighourhood species composition. New Phytologist 211: 455 463. Ó 217 The Authors New Phytologist Ó 217 New Phytologist Trust
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