Seagrass genotypic diversity increases disturbance response via complementarity and dominance

Transcription

1 Journl of Ecology 211, 99, doi: /j x Segrss genotypic diversity increses disturnce response vi complementrity nd dominnce A. Rndll Hughes 1 * nd John J. Stchowicz 2 1 Costl nd Mrine Lortory, Florid Stte University, 3618 Highwy 98, St. Teres, FL 3238, USA; nd 2 Deprtment of Evolution nd Ecology, University of Cliforni-Dvis, One Shields Avenue, Dvis, CA 9616, USA Summry 1. Genetic diversity, like species diversity, cn enhnce resistnce or resilience to perturtion. However, we know little out how disturnce intensity ffects this reltionship or wht mechnisms underlie the positive effects of genetic diversity. 2. We experimentlly tested the independent nd interctive effects of segrss genotypic diversity (two levels) nd disturnce (three levels) on segrss iomss in 2-yer field experiment. 3. Our results indicte tht genotypic diversity enhnces segrss resilience from experimentl iomss removl, ut only t the highest level of disturnce; in the sence of disturnce, monocultures out-perform polycultures over the short term. 4. Following recovery from the plnned experimentl disturnce, nturl mcrolgl loom cused loss of segrss shoots in our plots. In this cse polycultures lost fewer shoots thn monocultures (i.e. were more resistnt to the disturnce), nd this positive effect of genetic diversity persisted until the end of the experiment (1 yer in totl, including 6 months fter ll plots hd recovered to pre-disturnce densities). At the end of the 2-yer experiment, polycultures hd higher shoot density nd ove-ground iomss thn monocultures.. The positive effects of diversity on shoot density nd iomss were cused y oth trit-independent complementrity (TIC; due to differentil resource use mong clones) nd positive dominnce (due to one genotype chieving high density in oth monoculture nd polyculture). 6. Synthesis. Our results confirm tht genetic diversity, like species diversity, cn influence disturnce response nd does so vi similr mechnisms. They lso highlight tht over longer time frmes, these effects re likely to result from complex mix of dominnce nd complementrity mechnisms tht depend on the trits of the specific tx involved nd the response vriles of interest. Key-words: qutic plnt ecology, iodiversity, community genetics, compenstion, disturnce stility, ecosystem function, eelgrss, genotypic diversity, primry production, resilience, Zoster mrin Introduction The reltionship etween iodiversity nd stility hs long interested ecologists, generting lrge ody of empiricl nd theoreticl reserch. Although still controversil, the empiricl evidence suggests tht species diversity most often influences stility y reducing relized disturnce (Hughes et l. 27) vi vriety of mechnisms. For exmple, the insurnce hypothesis posits tht more diverse communities re more likely to contin tx cple of withstnding or surviving given disturnce tht cn compenste for those tht re more susceptile (Tilmn 1996; Ychi & Loreu 1999). In this cse, *Correspondence uthor. E-mil: rhughes@io.fsu.edu these tx re expected to increse in reltive undnce following disturnce, s is the cse in the smpling effect in experiments of iodiversity nd ecosystem function (Huston 1997; Tilmn, Lehmn & Thomson 1997). Additionlly, diversity my e ssocited with n incresed rnge of life-history or resource-use trits tht enhnce the response to disturnce vi complementrity or fcilittion (Mulder, Ulissi & Dok 21). To dte, the mjority of empiricl studies of the diversity disturnce reltionship hve focused on diversity t the level of species, yet the mechnisms thought to e responsile for diversity stility reltionships re not specific to ny prticulr txonomic level (Norerg et l. 21). The opertion of these mechnisms depends only on tx vrying in some Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society

2 446 A. R. Hughes & J. J. Stchowicz functionlly relevnt mnner; this sort of vrition is certinly not restricted to the species level, s studies show tht phenotypic vrition within species cn e s lrge s tht etween species (Bngert et l. 2; Shuster et l. 26). In ddition, genetic diversity hs demonstrted popultion- nd community-level ecologicl effects similr to those of species diversity (Hughes et l. 28). Although the numer of exmples is smll, the ville dt suggest tht genetic diversity cn lso influence popultion stility y reducing relized disturnce (Schmitt & Antonovics 1986; Boles, Thoendel & Singh 24; Hughes & Stchowicz 24; Reusch et l. 2) y incresing resistnce (decresed iomss loss) or resilience (recovery to pre-disturnce conditions). In mrine systems, two independent studies hve shown tht genotypic diversity in segrss (Zoster mrin) enhnces the resistnce nd resilience of this system to nturl disturnces (Hughes & Stchowicz 24; Reusch et l. 2), nd tht these positive effects of diversity cn e detected in nturl popultions of Zoster (Hughes & Stchowicz 29). However, these studies involved nturl, uncontrolled disturnces, leving open the question of how segrss genotypic diversity (nd genetic diversity, more generlly) influences system response cross rnge of disturnce levels. Additionlly, these studies were reltively short in durtion, spnning only single growing seson. In this pper, we exmine the effects of segrss genotypic diversity on segrss density nd iomss in response to experimentlly imposed disturnces over the short term (weeks to months) nd to lrger nturl disturnces over the long term (> 1 yer). We hypothesized tht genotypic diversity would hve positive effect on the response of segrss plots to experimentlly imposed disturnce. However, we expected tht these differentil responses to disturnce would e temporry, resulting in equivlent performnce etween monocultures nd polycultures t the end of longer period of time (2 yers). Eelgrss (Z. mrin, Zoster henceforth) is model system for testing the interction etween genotypic diversity nd disturnce for numer of resons. First, Zoster reproduces sexully s well s clonlly, generting considerle vrition in genotypic diversity t smll scles in nturl popultions (Reusch 21; Hughes & Stchowicz 29). In ddition, genotypic diversity in these popultions is importnt not only for the segrss itself, ut it lso influences the undnce nd diversity of orgnisms tht rely on it for hitt (Hughes & Stchowicz 24, Reusch et l. 2). Furthermore, segrsses re sujected to wide vriety of nturl nd nthropogenic disturnces, such s grzing (y invertertes, fishes nd irds), storm events, eutrophiction, ot grounding nd dredging (Short & Wyllie-Echeverri 1996; Orth et l. 26). These vried nd common disturnces represent mjor thret to segrsses, which re currently declining throughout their rnge (Wycott et l. 29). Understnding the role of segrss genotypic diversity in the response to nd recovery from disturnce is incresingly importnt for the conservtion of these vlule hitts. Mterils nd methods STUDY SYSTEM Bodeg Hror is locted c. 1 km north of Sn Frncisco By, CA, USA. It is c. 2km 2 mrine emyment tht is lrgely flushed on ech tidl cycle y centrl, dredged shipping chnnel. Zoster is common throughout the low intertidl nd sutidl regions of the hrour. This experiment ws conducted within n existing segrss ed on the south-western side of the hrour. Previous experimentl mnipultions of genetic diversity in Zoster eds (Hughes & Stchowicz 24; Reusch et l. 2) relied on field-collected clones tht were identified using moleculr mrkers, yet this process introduces numer of difficulties. First, the genotyping process is costly nd time-consuming. More importntly, however, it is difficult to find dequte numers of shoots of the sme clone to include ech clone in oth monoculture nd polyculture to llow inferences out mechnisms underlying diversity effects. Further, using field-collected shoots from different res mens tht genotypic effects my e confounded with locl environmentl cclimtion. To minimize these complictions, we propgted eight known genotypes in common grden 3-L outdoor flow-through mesocosms t Bodeg Mrine Lortories (see Hughes et l. (29) for detils on clonl propgtion). EXPERIMENTAL DISTURBANCE MANIPULATION We used shoots from the clones propgted in the lortory to conduct fctoril field experiment in which we mnipulted segrss genotypic diversity (one or six genotypes) nd disturnce (%, 33% or 66% of shoots clipped) nd monitored shoot density over time. Shoot density is non-destructive smpling technique, llowing us to ssess how results chnge over time while voiding the introduction of dditionl disturnce tht could oscure our disturnce tretments. Shoot density is positively correlted with ove-ground iomss in this system (R. Hughes, unpulished dt), so it is resonle mesure of iomss production. We lso destructively smpled ll plots t the end of the experiment. The genotypic diversity tretments tested in our experiment fll within the rnge found in nturl Zoster popultions in Bodeg Hror of 1 1 genotypes m )2 with men of 4.1 (Hughes & Stchowicz 29). Becuse the est wy to differentite etween mechnisms of complementrity nd selection effects is to compre genotype performnce in monoculture nd polyculture (Loreu & Hector 21; Fox 2), we plnted three monocultures (one for ech disturnce tretment) of ech of the eight genotypes used in the experiment. For the polyculture tretments, we rndomly generted eight different comintions of six genotypes nd plnted one replicte of ech per disturnce tretment; y chnce, one genotype ws present initilly in ll polyculture tretments. Thus we hd replictes of monocultures nd polycultures, ut no repliction of specific compositions within ech level of disturnce. We did this ecuse we hd limited numers of shoots ville for ech genotype, nd ecuse in this experiment we were most interested in whether monocultures differed from polycultures rther thn compring the performnce of specific genotypes or comintions of genotypes. However, t the end of the experiment, there ws no effect of disturnce on ny metric of performnce, nd we removed this fctor from the nlysis, giving us N = 3 per genotype comintion in the finl nlysis. We used six genotypes in our polyculture tretment, ecuse this diversity ws well within the rnge oserved in nturl popultions (i.e. we did not is our results y using n extremely high diversity) nd ecuse this Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

3 Genetic diversity enhnces stility 447 llowed us to hve high diversity plots tht vried in genotypic composition. All tretment plots egn with six trnsplnted shoots, regrdless of whether it ws monoculture or polyculture. Limittions on the numer of shoots ville per genotype necessitted these low initil densities; plots reched densities comprle to surrounding nturl segrss eds in the second yer of the experiment (see Results). We lso creted control plots tht received no trnsplnts to estimte nturl recruitment of shoots into re spce over the course of the experiment. In My 2 we clered 64.1-m 2 plots in 4 rows of 16 plots (seprted y t lest 2 m) in n existing segrss ed in Bodeg Hror. We then uried 16.2 cm high 41.3 cm wide 9.7 long continers into ech plot nd filled them with sieved sediment to exclude ll seeds nd elow-ground vegettion nd ensure tht there were no differences in the infunl inverterte community t the strt of the experiment. In preliminry trils, shoot production rtes did not differ inside versus outside continers (men = 1.1 shoots trnsplnt )1 month )1 inside continers vs..9 shoots trnsplnt )1 month )1 outside; t-test, P =.39). Tretments were ssigned to plots in rndomized complete-lock design, with two plots of ech disturnce diversity comintion nd four control plots per lock. Experimentl shoots were trnsplnted into the plots in June 2, nd shoot densities were monitored every 2 4 weeks for the durtion of the experiment (June 27). In Novemer 2, ll plots were surrounded y four 2-m tll pieces of PVC to prevent shoot removl y geese tht could confound interprettion of the experiment. We evluted the effects of genotypic diversity on the response to disturnce y pplying three levels of ove-ground iomss removl in Jnury 26 (8 months post estlishment). The removl of ove-ground iomss ws chosen specificlly to simulte grzing y geese, nturl nd significnt disturnce in Bodeg Hror (Hughes & Stchowicz 24), lthough ove-ground iomss removl is common thred mong mny segrss disturnces (Short & Wyllie-Echeverri 1996; Orth et l. 26). The specific disturnce levels correspond with vrition in goose grzing oserved in previous experiment (Hughes & Stchowicz 24). Although plots were still incresing in shoot density y vegettive propgtion t the time of the experimentl disturnce, we wnted to perform the disturnce tretment in n ecologiclly relevnt seson (i.e. when goose grzing typiclly occurs) nd efore the recruitment of new genotypes y seed (Mrch April) tht could potentilly compromise diversity tretments. In erly Jnury, we quntified shoot density in ech plot; these dt were then used to clculte the numer of shoots tht represented %, 33% or 66% of the ove-ground iomss. We hphzrdly selected the pproprite numer of shoots in ech plot nd removed iomss y mnully clipping shoots to the sediment surfce. In ddition to stndrdizing the mount of iomss removed within tretment level, our experimentl disturnce llowed us to void confounding non-consumptive effects (fertiliztion, sediment disturnce) tht re specific to herivores, since we wnted to ssess whether there is generl effect of diversity on disturnces tht cuses loss of ove-ground iomss. EXPERIMENTAL DISTURBANCE MANIPULATION: STATISTICAL ANALYSES We first ssessed the effectiveness of our disturnce tretment y conducting fctoril nov on the numer of shoots 2 weeks following the experimentl disturnce, with lock, diversity nd disturnce intensity s fixed fctors nd including ll possile interctions. We included pre-disturnce shoot density s covrite (see Hughes & Stchowicz 24); in these nd susequent nlyses incorporting covrites, our dt met the ssumption of homogeneity of slopes. To ssess the influence of diversity on recovery from disturnce, we performed repeted-mesures nov on ll smpling dtes during the recovery period (12 weeks), using shoot density 2 weeks following the disturnce s our covrite. There ws significnt diversity disturnce interction in the repeted mesures nlysis (Tle 1), so we exmined the effects of diversity t ech smpling dte (4, 6, 1 nd 12 weeks post disturnce) using plnned independent contrsts to compre monoculture nd polyculture performnce for ech disturnce level. Our nlysis did not meet the ssumptions of sphericity, so we used the Greenhous Geisser (G G) djustment to reduce our degrees of freedom when evluting time effects. Becuse there were not significnt interctions etween diversity nd or disturnce with time, we present dt from 6 weeks post disturnce to illustrte the effects of diversity on recovery from disturnce. In these nd susequent nlyses, we excluded one plot from our nlyses in which Zoster trnsplnts filed to estlish. NATURAL DISTURBANCE EVENT Twelve weeks following our experimentl disturnce (April 26), there were no longer ny differences in shoot density due to disturnce, diversity or their interction (Tle 1). Severl months lter (August 26), n unexpected loss of shoots occurred cross ll tretments. This shoot loss ws ssocited with nturl loom of the green mcrolg Ulv sp. in our plots nd the surrounding segrss eds of Bodeg Hror. The loom lsted from My to Octoer, ut reched its gretest intensity in lte June 26, peking t 4 kg wet weight m )2, the gretest iomss recorded during the 4-yer period from 24 to 28 (Olyrnik 28). Becuse of the oserved shoot Tle 1. Results of sttisticl nlyses of the interction etween disturnce nd genotypic diversity. Disturnce (d.f. = 2) Diversity (d.f. = 1) Disturnce Diversity (d.f. = 2) Time since disturnce F P F P F P Covrite 2 weeks < weeks 4 weeks weeks 6 weeks weeks 1 weeks weeks 12 weeks weeks Overll (repeted mesures) Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

4 448 A. R. Hughes & J. J. Stchowicz loss following pek Ulv undnce nd the documented negtive effects of high Ulv iomss on segrsses (Huxwell et l. 21; McGlthery 21, Olyrnik 28), we treted this lgl loom s nturl disturnce nd continued running the experiment to cpture the effect of genotypic diversity on the Zoster response to this disturnce. In June 27, we mesured finl shoot density nd hrvested the segrss from ll plots. We then quntified distnce etween shoots, nd shoot, root nd rhizome iomss from hphzrdly selected clones (i.e. multiple shoots connected y single rhizome) from ech plot. We lso genotyped these smpled clones to ssess the performnce of the plnted genotypes nd determine finl genotypic composition. Becuse of the higher proility of not smpling genotype tht ws ctully present in polyculture thn in monoculture (due to the lower initil reltive undnce of component genotypes in polyculture), we smpled polycultures with greter intensity: we smpled 48 clones from ech polyculture nd 24 clones from ech monoculture, representing 26 1% of the shoots in ech plot (verge = 8%). The remining segrss iomss ws divided into ove- nd elow-ground portions, dried t 6 C for t lest 48 h, nd weighed to determine iomss. NATURAL DISTURBANCE EVENT: STATISTICAL ANALYSES We rn fctoril nov on shoot density fter the Ulv loom dissipted (7 Septemer 26) with shoot density prior to the decline (26 July 26) s covrite, including lock, diversity, disturnce intensity nd ll possile interctions s fixed fctors. We lso quntified Ulv iomss in July 26 nd rn fctoril nov s ove to determine if Ulv vried y disturnce or diversity tretment. We were unle to relily smple density in our plots in August 26 due to lck of sufficiently low tides during this month. To evlute differences etween monocultures nd polycultures in shoot density, ove-ground iomss, elow-ground iomss, root : rhizome iomss rtio, nd distnce etween shoots t the end of the experiment, we rn fctoril novs on ech response vrile individully with lock, diversity, disturnce intensity nd ll possile interctions. Neither the experimentl disturnce tretment nor ny of its interctions ffected ny of the response vriles during this period (F.78, P.47), so we dropped this fctor nd its interctions from our nlyses. Thus, there were three replictes of ech monoculture nd polyculture comintion for this portion of the nlysis. To exmine finl genotypic composition nd reltive undnce, we collected tissue smples from ech of the hphzrdly selected clones in ech plot t the end of the experiment. The smples were frozen following collection nd lter extrcted using modified CTAB protocol, mplified vi polymerse chin rection, nd genotyped t five DNA microstellite loci specific to Z. mrin (see Hughes & Stchowicz 24 for Methods). Shoots differing y zero or one lleles were considered of the sme genotype in our nlyses (Arnud-Hond et l. 27). GenClone 2. ws used to clculte finl genotypic diversity. IDENTIFYING DIVERSITY MECHANISMS To test whether polyculture performnce differed on verge from tht expected sed on performnce of the component genotypes in monoculture, we compred oserved nd expected polyculture vlues using pired Student s t-tests t three occsions in our experiment when there ws significnt effect of genotypic diversity: (i) following our experimentl disturnce; (ii) following shoot loss during the mcrolgl loom; nd (iii) t the end of the 2-yer experiment. We clculted expected polyculture vlues sed on the initil genotypic composition of ech polyculture, ssuming equl reltive undnces (i.e. monoculture vlues were divided y six, the originl numer of trnsplnts, nd then summed for ech genotype in prticulr polyculture comintion). A significnt difference etween oserved nd expected vlues indictes overyielding hs occurred. To further differentite mong diversity mechnisms, we used stndrd methods (Fox 2) to prtition vrition in polyculture shoot density nd ove-ground iomss t the end of the experiment mong three mechnisms: TIC, dominnce nd trit-dependent complementrity. TIC (equivlent to complementrity sensu Loreu & Hector 21) results when genotypes yield higher iomss in mixture thn expected, regrdless of solute monoculture iomss. TDC nd dominnce (together equivlent to selection sensu Loreu & Hector 21) occur when prticulr genotype domintes polycultures, either t the expense of other genotypes (dominnce) or in ddition to increses in other genotypes (TDC). Becuse there were no independent or interctive effects of disturnce t this time, we used the three disturnce levels s replictes for ech prticulr genotypic comintion. We conducted seprte novs for iomss nd density to determine whether the diversity mechnisms differed in their effect size. Results EXPERIMENTAL DISTURBANCE MANIPULATION There ws generl increse in shoot density over the 2-yer experiment, regrdless of disturnce level or genetic diversity (Fig. 1); the finl shoot density of 21 ± 14.7 shoots.2 m )2 (men ± SE) is similr to tht found in undistured plots in Numer of shoots per.2 m ! Monoculture Polyculture Unmnipulted Jun 2 Jul 2 Aug 2 Sept 2 Oct 2 Nov 2 Dec 2 Jn 26 Fe 26 Mr 26 Apr 26 My 26 Jun 26 Jul 26 Aug 26 Sept 26 Oct 26 Nov 26 Dec 26 Jn 27 Fe 27 Mr 27 Apr 27 My 27 Jun 27 Dte Fig. 1. Segrss shoot density over the course of the experiment. Grey dimonds represent monocultures; lck squres re polycultures. Open circles re from n unmnipulted nturl segrss ed in nery experiment (Olyrnik 28). Error rs represent 1 SE. () The experimentl disturnce ws pplied in Jnury 26. There ws significnt interction etween diversity nd disturnce during the 12-week period highlighted in the ox (see Fig. 2). () A nturl loom of the mcrolge Ulv egn in My 26 nd ended in Octoer 26, resulting in loss of shoots cross ll plots (nd in nery unmnipulted res). Shoot density in our experimentl plots ws comprle to the nturl shoot density throughout the second yer of the experiment. Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

5 Genetic diversity enhnces stility 449 the surrounding eelgrss ed in seprte study (Olyrnik 28). Prior to the experimentl disturnce there ws no effect of diversity tretment on shoot density, with plots of on verge c. 4 shoots per continer. Our experimentl disturnce in Jnury 26 ws effective, resulting in significnt decline in the numer of shoots per plot 2 weeks following the disturnce s clipping intensity incresed, regrdless of diversity (disturnce F 2,22 = 42.19, P <.1; Fig. 2). Not surprisingly, given tht we controlled the numer of shoots clipped, there ws no vrition in shoot density due to diversity t this time (diversity disturnce F 2,22 =.14, P =.71; Fig. 2). However, there were significnt diversity effects on disturnce resilience, mesured s shoot re-growth following Chnge in shoot density fter disturnce Weeks to pre-disturnce density () Monoculture Polyculture Expected polyculture () 4 (c) Disturnce (% of shoots clipped) Fig. 2. Clonl diversity nd disturnce interct to influence segrss shoot density. Open rs re monocultures; lck rs re polycultures; htched rs re expected vlues for polycultures. Error rs represent 1 SE. () Segrss response 2 weeks fter n experimentl disturnce. Disturnce response is shown s the difference in the numer of shoots 2 weeks fter the disturnce s compred to immeditely prior to the disturnce. As expected, no disturnce plots gined shoots over this period, wheres high disturnce plots lost shoots, regrdless of genotypic diversity. () Segrss response 6 weeks fter n experimentl disturnce. Disturnce response is shown s the difference in the numer of shoots t 6 weeks nd 2 weeks post-disturnce. Monoculture response did not differ cross the disturnce grdient, ut polycultures experienced net shoot loss under no disturnce nd net shoot gin t the highest level of disturnce. The expected verge polyculture performnce sed on the men performnce of component monocultures differed from oserved polyculture performnce t oth zero nd high disturnce. (c) Time to recovery to pre-disturnce shoot density. Differences in shoot re-growth following disturnce (pnel ) led to vrition in the numer of weeks until recovery to pre-disturnce density in the high disturnce tretment: monocultures took lmost twice s long to recover s polycultures. disturnce (repeted-mesures nov: diversity disturnce F 2.22 = 3.64, P =.4, see Tle 1 for full sttistics), nd these effects persisted for 1 weeks (time effect: G G F 46,42 =1.16,P =.31). Our experimentl disturnce in Jnury 26 coincided with sesonl period of low growth (see, e.g. similr period of sttic shoot density in Jnury 27; Fig. 1), most likely due to light limittion (Dennison & Alerte 1982). Polycultures nd monocultures in the no-disturnce tretment differed in shoot growth during the recovery period (independent contrst, F 1,22 =.73, P =.2): polycultures experienced slight net decrese in shoots, despite not eing clipped. While monocultures exhiited no net chnge in shoot numers (Fig. 2). Shoot mortlity nd re-growth were equivlent mong diversity tretments t intermedite disturnce (independent contrst, F 1,22 =.4, P =.47), nd there ws trend for polycultures to gin more shoots thn monocultures t the highest level of disturnce (independent contrst, F 1,22 = 3.4, P =.9). The oserved polyculture chnge in shoot density ws less thn expected sed on performnce of clones in monoculture t zero disturnce (pired t-test, P =.6) ut did not differ from expected t intermedite disturnce (pired t-test, P =.14). In contrst, polycultures hd greter increses in shoot density thn expected t the highest level of disturnce (Fig. 2, pired t-test, P =.1). The difference in the rte of re-growth etween monocultures nd polycultures t high disturnce cused the recovery time to pre-disturnce densities to vry y diversity nd disturnce (nov: diversity disturnce F 2.22 =3.23,P =.); polycultures recovered fster thn monocultures t high disturnce (independent contrst, F 1,22 = 4.94, P =.4; Fig. 2c). NATURAL DISTURBANCE EVENT Diversity ffected shoot density in the presence of the mcrolge Ulv sp. Prior to nd during the erly stges of the loom (April July 26) there ws no difference in shoot density etween monoculture nd polyculture for ny disturnce tretments (Fig. 1; diversity F 1, , P.19). Differences in shoot density were not significnt until the end of the loom in lte summer (diversity F 1,3 =4.82,P =.4), fter period of shoot loss during time of yer when expnsion would normlly occur (Fig. 1). All plots lost shoots during this period, yet polycultures lost smller percentge of shoots thn monocultures (Fig. 3). There ws no effect of clipping tretment or diversity on the iomss of Ulv during the loom (F 1.4, P.22), so we ttriute the differences in shoot density fter the loom to effects of genotypic diversity on response to the loom. However, it is lso possile tht diversity effects in the sence of disturnce simply tke time to mnifest themselves, nd the timing of the loom coincided with sufficient time elpsing to rech shoot densities chrcteristic of nturl eds (Fig. 1). Regrdless, the percentge of shoots remining in polycultures following the loom ws significntly higher thn tht expected sed on component monocultures (pired t-test P =.6). Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

6 4 A. R. Hughes & J. J. Stchowicz Percentge shoots remining Monoculture Polyculture Expected Fig. 3. Segrss genotypic diversity increses the response to nturl disturnce. Open rs re monocultures; lck rs re polycultures; htched rs re expected vlues for polycultures. Error rs represent 1 SE. The percentge of shoots remining fter period of shoot loss in summer 26. Significntly more shoots survived this nturl disturnce event in polycultures thn in monocultures. In ddition, oserved polyculture performnce exceeded tht expected sed on component monocultures. Unlike the trnsient diversity effect in response to the controlled shoot removl, the positive effect of genotypic diversity following the Ulv loom persisted until the end of the experiment in June 27, nerly 1 yer fter the end of the loom (monoculture men(se) shoots.2 m )2 = 161.9(16.3); polyculture men(se) shoots.2 m )2 = 243.6(16.7); F 1,39 = 12.31, P =.1; Fig. 1). Additionlly, there ws corresponding difference in ove-ground iomss etween polycultures nd monocultures (F 1,39 = 8., P =.7; Fig. 4). In contrst, elow-ground iomss did not differ etween monocultures nd polycultures (F 1,39 =2.93, P =.9; Fig. 4). Despite the similrity in overll elowground iomss, the lloction to root or rhizome tissue differed, with polycultures hving greter proportion of root tissue (F 1,39 = 4.8, P =.3; Fig. 4c). Also, the verge distnce etween shoots long rhizome segment ws lower for polycultures thn monocultures (F 1,39 = 6.46, P =.1; Fig. 4d), indicting tht shoots were pcked more closely together in polycultures. This result ws lrgely due to the extremely low shoot spcing of one dominnt genotype (men(se) distnce etween shoots = 4. ± 2.1 mm) reltive to the other genotypes (mens rnging from 2.3 to 77.2 mm etween shoots). Oserved polyculture vlues were consistently different thn expected for ll responses except elow-ground iomss (pired t-tests P.1; Fig. 3). Becuse we destructively smpled the experimentl plots t this time nd were le to estimte the reltive contriutions of different genotypes to finl density nd iomss, we could ssess the degree to which dominnce of prticulr genotypes vs. complementrity mong genotypes were responsile for the oserved diversity effects. Prtitioning the effects of diversity shows tht oth mechnisms ply role, ut their reltive importnce differs for density nd iomss. The positive net diversity effect on density ws due to positive TIC, positive dominnce nd positive TDC (Fig. c). Although the strength of these mechnisms did not vry overll (F 2,14 = 2.42, P =.12), exmining the different polyculture comintions seprtely revels tht oth dominnce nd TIC cn e quite strong (Fig. d). The strong dominnce, when it occurred, ws driven y the high shoot production of single genotype, 8 (Fig ; lelled white in Hughes et l. 29) tht lso performed well in monoculture (Fig. ). However, genotype 8 ws not le to dominte ll polyculture comintions, s illustrted y the vrying strength of dominnce (dominnce = in three out of eight polycultures tht hd this genotype; Fig. d). Furthermore, dominnce did not ply n importnt role in the diversity effect on ove-ground iomss (Fig. g); rther, strong nd consistently positive TIC creted this positive diversity effect (Fig. g,h). The numer of genotypes in polycultures remined significntly higher thn monocultures t the end of the 2-yer experiment (polyculture men(se) genotypes = 7.3(.4), monoculture men(se) genotypes = 2.(.4); F 1,39 = 12.32, P =.1). Reltively few new clones were identified in our finl genetic smpling (men(se) new clones per plot = 2.7(.4); mx = 7.) nd, more importntly, these were never () Aove-ground iomss (g) Monoculture Polyculture Below-ground iomss (g) Monoculture Polyculture (c) (d) 1. e 7 h h.9 d d 6 g Monoculture Polyculture Expected Monoculture Polyculture Expected Root to rhizome rtio Expected () Shoot spcing (mm) Expected Fig. 4. Positive effects of segrss genotypic diversity fter two yers. Open rs re monocultures; lck rs re polycultures; htched rs re expected vlues for polycultures. Error rs represent 1 SE. () Averge plot ove-ground iomss. () Averge plot elow-ground iomss. (c) Rtio of root iomss to rhizome iomss. (d) Averge distnce etween 1 nd shoots on 24 (monoculture) or 48 (polyculture) rhizome segments per plot. Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

7 Genetic diversity enhnces stility 41 Finl shoot density Percentge of finl density Effect size Effect size () M1 M2 M3 M4 M M6 M7 M8 M1 M2 M3 M4 M M6 M7 M8 Monoculture tretment () P1 P2 P3 P4 P P6 P7 P8 (c) (d) TIC Dominnce TDC Finl iomss (g) Percentge of finl iomss 2 (e) (h) 1 1 P1 P2 P3 P4 P P6 P7 P8 Polyculture tretment (g) TIC Dominnce TDC 2 TIC Dominnce TDC Diversity mechnism 1 P1 P2 P3 P4 P P6 P7 P8 P1 P2 P3 P4 P P6 P7 P8 Polyculture tretment Fig.. Genotypic composition of monocultures nd polycultures fter 2 yers. Segrss () shoot density nd (e) ove-ground iomss in June 27 in monoculture tretments. Segrss () shoot density nd (f) ove-ground iomss in June 27 in polyculture tretments. Shding indictes the reltive undnce of component genotypes to finl density. Genotypes correspond to the following colours in Hughes et l. (29): 1 (red); 2 (green); 3 (yellow); 4 (ornge); (purple); 6 (lue); 7 (grey); 8 (white). (c, g) Overll contriution of TIC (closed rs), dominnce (grey rs), nd TDC (open rs) to (c) density nd (g) ove-ground iomss. Letters indicte significnt differences (P.) ccording to Tukey s post hoc tests. (d, h) Reltive importnce of TIC, dominnce, nd TDC in polyculture tretments for (d) density nd (h) ove-ground iomss. (f) very undnt (Fig.,f; men(se) percentge of totl = 6.(1.6)%). This result is consistent with our oservtion of very low seedling recruitment into our control plots over the course of the experiment (men(se) finl shoot density = 16.1(.3), dt not shown). Though genotypic diversity differed from tht initilly plnted t the end of the experiment, we continue to refer to the initil monoculture nd polyculture tretments for consistency. Discussion Segrss genotypic diversity enhnced recovery from n experimentl disturnce in our study, lthough these effects were only evident t the highest disturnce intensity. The interction etween diversity nd disturnce intensity in this study hd not een demonstrted previously, illustrting the importnce of controlled, experimentl mnipultions in order to completely understnd the reltionship etween diversity nd disturnce. In prticulr, the loss of shoots with high diversity in the sence of disturnce is puzzling nd hrd to explin. The effect ws smll in mgnitude, reltively short-lived (<12 weeks), nd could e consequence of mesurement during time of yer when growth is slow nd sesonl declines in density re common (Fig. 1). We did not oserve comprle effect in polycultures during the rest of the experiment or in ny other experiment we conducted (e.g. Hughes & Stchowicz 24). If verified, underyielding y polycultures in the sence of disturnce could hve importnt Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

8 42 A. R. Hughes & J. J. Stchowicz conservtion nd mngement implictions nd deserves further ttention. The positive diversity effect t high disturnce, comined with the oserved increse in disturnce response y polycultures to nother high-intensity disturnce ( nturl loom of the mcrolge Ulv), corroortes the positive effects of diversity suggested y previous nturl (i.e. unmnipulted) disturnces in this system (Hughes & Stchowicz 24; Reusch et l. 2). In ddition, sttisticl prtitioning of the diversity effect indictes tht oth positive TIC nd positive dominnce (rther thn negtive s in Reusch et l. 2) cn e importnt for these effects. However, these sttisticl methods cnnot specify the iologicl mechnisms underlying diversity effects. One cvet to the results of our experimentl disturnce mnipultion is tht shoot density ws elow mient (leit not incresing) when we imposed the disturnce, potentilly influencing the results. However, the rodly consistent effects of genetic diversity on disturnce response etween the experimentl disturnce nd nturl disturnce (which occurred when shoot density ws ner mient) even though they my rise y different mechnisms increse our confidence tht these effects re not dependent on initil shoot density. It is interesting to note tht the effects of diversity persisted for much longer, i.e. until the end of the experiment (1 yer, Fig. 4), when disturnce occurred t time when shoot density ws ner mient (Fig. 1). There re two potentil explntions for this pttern. First, genotypic diversity could hve much stronger nd more prolonged impct on shoot density in response to Ulv thn in response to our experimentl disturnce, due either to differences in the intensity or the mechnisms of disturnce. Alterntively, stronger interctions mong genotypes once nturl densities were reched during yer 2 could hve resulted in stronger diversity effects, consistent with previous studies suggesting tht diversity effects due to complementrity strengthen over time (Crdinle et l. 27, Stchowicz et l. 28). We cnnot currently rule out either of these explntions. Becuse the experimentl clipping process overrode ny inherent differences mong shoots in their ility to withstnd disturnce, the positive impct of diversity in response to our mnipultion must hve een due to incresed shoot production following disturnce. This increse in shoot production in polyculture is consistent with previous studies of segrss genetic diversity (Reusch et l. 2), s well s mnipultions of species diversity (Stchowicz, Bruno & Duffy 27). In contrst, the positive diversity effect in response to Ulv ws t lest in prt due to polycultures losing smller percentge of shoots thn monocultures (Fig. 3). This oserved decrese in the numer of shoots cme during sesonl period of shoot gin, so the effects of Ulv on Zoster were likely greter thn indicted y the decline in Fig. 1. In fct, n experimentl Ulv removl study conducted less thn 1 km from our study site t the sme time found tht mient Ulv iomss of 4.39 kg m )2 cused 8% reduction in shoot density in summer 26 (Olyrnik 28, Fig. 1). Thus, the Ulv iomss of 6.2 kg m )2 found in our plots cn resonly e considered high-intensity disturnce. When monoculture nd polyculture responses differed over the course of the experiment, the oserved polyculture response ws consistently greter thn tht expected y the verge performnce of ech of the genotypes in monoculture, indicting tht overyielding occurred. At the end of the experiment, ll three diversity mechnisms contriuted positively to the overll effect on shoot density (Fig. c), wheres TIC lone cused diversity effects on ove-ground iomss (Fig. g). When it occurred, the strong positive dominnce effect on shoot density ws correlted with high-reltive undnce of single genotype (genotype 8). This genotype exhiited the highest shoot density in monoculture in this experiment, nd lso hd the highest rte of shoot production in seprte lortory experiment (Hughes et l. 29). Not only is its shoot production rte high, ut its reltive lloction to elowground iomss, nd specificlly root iomss, is lso high (Hughes 26; Hughes et l. 29); this higher lloction to elow-ground reserves my llow it to withstnd disturnces nd compenste for more susceptile genotypes (i.e. the insurnce hypothesis; Tilmn 1996; Ychi & Loreu 1999). In contrst to its high density, the totl ove-ground iomss of genotype 8 is equivlent to tht of the other genotypes (Fig. e; Hughes et l. 29), perhps explining the lck of positive dominnce for ove-ground iomss in this experiment. Strong, positive TIC in shoot density is not s esily explined y the trits of individul genotypes, ut it does pper to e correlted with the persistence of genotype 4 in polyculture (e.g. polyculture (P) 4, P, P6, P7; Fig.,c). Genotype 4 differs considerly from the other genotypes in its reltive rtes of nutrient uptke: it hs the highest rte of root mmonium uptke nd the lowest rte of lef nitrte uptke of ll of the genotypes used in the experiment (Hughes et l. 29). The ssocition of genotype 4 with positive TIC suggests tht vrition in resource utiliztion could drive these density effects. Differences mong genotypes in resource use (Hughes et l. 29) could lso contriute to complementrity effects on polyculture iomss. However, it is difficult to scrie complementrity in iomss to the trits of ny one genotype ecuse of its consistent strength cross replictes of different genotypic composition. The potentil generlity of strong, positive dominnce nd complementrity mong genotypes is uncler, s few studies of the ecologicl consequences of genetic diversity hve exmined finl genotypic composition to differentite mong potentil mechnisms of diversity effects (Hughes et l. 28). Even within this single experiment we found tht the reltive importnce of prticulr mechnisms vried considerly depending on the comintion of genotypes in polyculture (lso see Hughes, Best & Stchowicz 21) nd the response vrile considered. Nonetheless, the pprent contrst etween the positive dominnce found here nd the negtive selection effects documented in similr system (Reusch et l. 2) could e due to the longer experimentl durtion of our study llowing time for interctions mong genotypes to lter reltive undnces. Regrdless of mechnism, our finding tht segrss genotypic diversity increses the response of this system to controlled nd nturl disturnce dds to the grow- Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43

9 Genetic diversity enhnces stility 43 ing consensus of the importnce of diversity for ecosystem stility (Hughes et l. 27, Stchowicz, Bruno & Duffy 27). It lso highlights tht these effects my tke time to mterilize nd re likely to come from mix of dominnce nd complementrity effects tht depend on the trits of the specific tx involved. Acknowledgements We would like to thnk K. Aquilino, S. Attoe, R. Best, N. Bosch, M. Brcken, J. Byrnes, L. Crney, K. Edwrds, M. Ferner, A. Fish, K. Hmmond, J. Hos, D. Kimro, A. Lrson, B. Miner, E. Mullen, E. Mullney, T. Ng, S. Olyrnik, P. Reynolds, K. Selheim, C. Sorte nd B. Steves for help in the field. A. Fish nd T. DiMrco provided invlule ssistnce in the lortory. S. Olyrnik provided dt for the unmnipulted plots in Fig. 1. R. Best nd two nonymous reviewers provided criticl comments tht improved the mnuscript. This work ws supported y NSF grnt OCE to J.J.S. nd A.R.H. References Arnud-Hond, S., Durte, C.M., Alerto, F. & Serro, E.A. 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Proceedings of the Ntionl Acdemy of Sciences, 94, Wycott, M., Durte, C.M., Crruthers, T.J.B., Orth, R.J., Dennison, W.C., Olyrnik, S., Clldine, A., Fourquren, J.W., Heck Jr, K.L.., Hughes, A.R., Kendrick, G.A., Kenworthy, W.J., Short, F.T. & Willims, S.L. (29) Accelerting loss of segrsses cross the gloe thretens costl ecosystems. Proceedings of the Ntionl Acdemy of Sciences USA, 16, Ychi, S. & Loreu, M. (1999) Biodiversity nd ecosystem productivity in fluctuting environment: the insurnce hypothesis. Proceedings of the Ntionl Acdemy of Sciences USA, 96, Received 3 June 21; ccepted 29 Octoer 21 Hndling Editor: Ro Brooker Ó 21 The Authors. Journl of Ecology Ó 21 British Ecologicl Society, Journl of Ecology, 99, 44 43