9505, 2300 RA Leiden, The Netherlands Leiden,The Netherlands 9505, 2300RA 4 Netherlands. 3 2 Netherlands. The 50,6700ABBox Wageningen, 1 Olga Kostenko plants arthropodson inthe focal density of field the and metabolites plant secondary of concentration the on diversity plant of Effects Handling Editor:Matthew Heard Article type Date:20-Oct-2016 Accepted :18-Aug-2016 Received Date This article isprotected Allrights bycopyright. reserved. 10.1111/1365-2745.12700 version andtheVersi this between differences through the copyediting, typesetting, pagination forpublication accepted This articlehasbeen E-mail: [email protected] +31(0)317-473675 Fax: (0) 317-473632 Telephone: +31 * Correspondence: Running headline: Present address:Courbois TheNetherlands. 230,6700AEWageningen, UR, P.O.Box RIKILT -Wageningen Netherlands Institute of Ecology (NIOO-KNAW), AcceptedInstitute ofBiology, Section Plant Ecologyand Phytochemistry, Leiden University,Box PO Article : Standard Paper Paper :Standard 1* , Patrick P. J. , Patrick P.J. Mulder Diversity effects on plant chemistry andinsects chemistry onplant effects Diversity Olga Kostenko Olga Flora & Fauna Expert, Eshofweg 16, 6717 LWEde, The Eshofweg 16,6717 & FaunaExpert, Flora 2 , Matthijs Courbois and undergone full peer review buthasnotbeen review undergone fullpeer and on of Record. Please cite of Record. Please on and proofreading process, which may lead to
Department Ecology, ofTerrestrial PO 1,3 and T.Martijn Bezemer
this article this as doi: 1,4 on reproductive focal plants wasnotsignififocal plants on reproductive plan withincreasing vegetative stage decreased planting,yearsafter the 3. Two diversity effects. plantstodi alkaloids, PAs)ofthefocal communities aswellthegrowth,andprima the structuralcharac recorded vegetation. We years. plantswe Focal for three or 9species) in experimental plant communities that were maintained at different ofdiversity levels (1, 2,4 In arthropodcommunitiesonfocal 2. field, wesampled the unresolved. by surrounding plantsremains the diversity hostplantquality by ofthe or ar diversity onthe theeffectsofplant extent towhat andquality plant.However, wellasthesize thatfocal plantas of insects onafocal comm plant thesurrounding 1. Thediversity of Summary This article is protected by copyright. All rights reserved. copyright. This articleis protectedby plant. this to associated insects individualplants grplant quality of effects ofthene conditionstheeffects under natural 4. arthropod communitiesonfocalplantswerenot diversity. Structural equation andconcentration the foliar N ofvegetativeplantsfocal decreased withincreasing plant the diversity ofth by significantly affected ofbothvege Thesize community. neighbouring
AcceptedSynthesis. Article Plant qualitygreatlycan influence insect preferenceand performance. However, modelling revealed the effects that ofplant diversity onthe abundance of arthropods onfocalpl abundance ofarthropods sentangle potential the mechanisms causing the owing in those communities on the abundance of thosecommunitiesontheabundance in owing e neighbouring community, butthelevelsofPAs e neighbouring cantly affected by the diversity of the thediversity by affected cantly thropod community on a focal plant are mediated aremediated plant onafocal community thropod re also plantedinplotswithout surroundingre also ighbouring plant community can overrule the ighbouring canoverrule plantcommunity teristics of each of the surrounding plant ofthesurrounding ofeach teristics ry and secondary chemistry (pyrrolizidine chemistry (pyrrolizidine ry andsecondary unity can directly affect the abundance of abundance the affect unity directly can t diversity, while the abundance of arthropods ofarthropods abundance whilethe t diversity, mediated by changes in plant quality. inplantquality. changes by mediated tative and reproductive focal plantswasnot focal andreproductive tative
Jacobaea vulgaris Jacobaea ants that werestillinthe ants that plantsgrowing plants onthe insect associated communities toa focal plant are mediatedchanges bythe in communities (Awmack & Leather 2002). However, greatlyquality can influencetheinteraction availability andspace(Crawl ofnutrients,light effects maycompetition between result from the focal plant that and alters itsneighbours the secondary chemistry; Bart and (primary andquality size suchasplant plant, of afocal characteristics influence Barbosa in (reviewed effects associational host plantlocationandselectionofin In communities, thepresenceandidentity plant Introduction quality, Plantspeciesrichness. Key-words: This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedthe natural enemies herbivores, ofthe such as parasitoids ( susceptibility hypothesis In of Root 1972). contrast, theabundance withplantdiversity ( related negatively Kostenko (Bezemer community insect and its importantfactorthat also bean community can Apart fromthe neighbouring identity of plants, the diversity the neighbouring of plant neighbourin the orby focal plantquality Article et al. Biodiversity, Insect community, 2012;Wäschke , Atsatt & O on & Bowers 2006; Temperton on & 2006;Temperton Bowers et al. et al ʼ Dowd 1976), as well as the abundance and diversity of anddiversity aswelltheabundance 1976), Dowd 2015). Specialist herbivore loads on a focal plant may be a focalplantmay be herbivoreloadson 2015). Specialist associational resistancehypothesis associational . 2004;Scherber g itselfremainsunresolved. community generalist herbivores on a focal plant ( focal plant herbivoresona generalist sect herbivores. These effects are called called are effects These herbivores. sect et al. between plantsandtheirmultitrophicinsect Jacobaea vulgaris Jacobaea ey 1997). In turn, changes in plantnutritional In in 1997). turn, changes ey influences interactions between a focal plant a between interactions influences 2009). Neighbouring plants canalso plants 2009).Neighbouring of neighbouring plants whetherthe
et al. et al. enemies hypothesis 2007;Broz 2006; Unsicker , Phytochemistry, Plant effects of neighbouringeffects of can greatly can greatly influence , Tahvanainen & , Tahvanainen et al. et ,
Root 1973),is et al.
2010).These associational 2006; intraspecific variation inplantqualityintraspecific variation (Bukovinszky the by affected significantly is aplant with associated community and predator herbivore the cultivars thatand differ chemical innutritional quality,composition shownthat have of the fiel monospecific understood. However, plant quality ininfluencing performe have been majority ofthosestudies insects(revi ofherbivorous and performance intheplant,is compounds primary andsecondary thathostplantqualit Many shown studieshave 2000;CastagneyrolWhitham & 1983;White (Kareiva composition ofthelocalpoolinsectsthatcoul apparency the affects vegetation, thatdirectly ofplantdiversity,including these effects predictedmore diverse tobecommunities. higher in plant Several factors maycontribute to This article is protected by copyright. All rights reserved. copyright. This articleis protectedby off hypothesis by thedi influenced a plantcanbe al. ofsecondary affect theexpression neighbouringcommunity.s plant itwas Recently, plan a focal Infield, thenutritionalquality of the open question. andherbivore communities predator innatural and hence diverse plant isan communities varia intraspecific extend what
Accepted that theoriespredict ecological 2015).Three Article (Herms & Mattson 1992)statesthatpl (Herms& aboveground insectsinnatu tion inplant quality determines ofinsect the composition versity of the plantcommunity. The versity ofthe metabolites in focal plants (Mraja plants(Mraja infocal metabolites d experiments, for example with forexample experiments, d the structure or height oftheneighbouring orheight the structure ewed in Awmack & Leather 2002). The vast Thevast 2002). Leather & Awmack in ewed d in controlled environments and theroleof environmentsand d incontrolled of the focal plant as well as the size and size as wellthe focalplant of the y, characterised by the concentration of y, concentration the by characterised the concentrationofse d subsequently ‘spill over’ tothefocalplant ‘spill over’ d subsequently t can be influenced by the diversity ofthe the by t canbeinfluenced an essential factor influencing preference influencing preference anessentialfactor hown, for example, thatplantdiversity can hown, forexample, et al. ants willallocatemoreresources to et al. ral communitiesislesswell
2008;Poelman 2013; Moreira 2013;Moreira et al. condary metabolitesin condary growth-defence trade- growth-defence Brassica oleracea Brassica 2011; Wäschke et al. et et al 2009).To . 2016). et et
reproductive stage are more importantforplant more are reproductive stage ofaplant development during theontogenetic showed meta-analysis A recent plantdiversity. withincreasing focal plantshouldincrease herbivory, th host plants.Hence,towithstand asspecialis specialist togeneralist dominated, predicts that withincreasing plant diversity, communities herbivore willchange from plants. lowdefended generalists favour plantswithhigh prefer specialists whereby generalist ratioof todependonthe expected theconcentration Meijden 1996),statesthat (Eisenhauer competition fornutrients, water and and light defenceplant inmore diverse communities as increased diversitywill lead plant toincreased This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted compounds. secondary plant whycompounds, andthismay reprod alsoexplain of flowering plants toinsect herbivores increased willlead to levels defence of plant Baldwi& (Karban theory Induced plantdefence orotheravailabl to theprovisionofnectar andthepres size to insectsduetheincreased reproduc 2010).Moreover, &2009; Barton Koricheva (Rhoades defended whybetter they are explain Article et al. 2009). In contrast, the that levels of secondary plant chemicals generally increase increase generally chemicals plant ofsecondary levels that The resource concentration hypothesis concentration The resource specialist-generalist dilemma hypothesis e resources (Rhoades & Cates 1976; Feeny 1976). Feeny 1976). & Cates1976; (Rhoades e resources concentrations of defence compounds while ofdefence concentrations of secondary plant compounds in aplantis plantcompounds of secondary and specialist herbivores in the community, herbivoresinthe and specialist e concentration of plant defence compoundsina ofplantdefence e concentration hence to reduced growth plants toreducedhence focal of ts prefer monospecifi ts prefer (Barton & Koricheva 2010). Plants intheir 2010). &(Barton Koricheva 1979; Agrawal 2004; Lankau Agrawal & 2004; Kliebenstein 1979; ence of inflorescences, and more attractive due more attractive and ofinflorescences, ence fitness than vegetative plants andthismay thanvegetative plants fitness n 1997) predicts that the increased exposure exposure 1997) predictsthattheincreased n uctive plants will express higher levels of higher levels uctive plantswillexpress tive plants are generally more apparent apparent generally more tive plantsare
c communities oftheir c (Root1973) vander insects, incontrast, deterred are them not tolocate by PAsbututilise hostsorsequester PAs microorganisms, andhumans(review mammals thatare toxi compounds containing secondary 2013). duetothebrig apparent tall andarehighly 1979).Th der Waals-Kooi Van & Van derMeijden of or whenthesize plant hasbeendamaged flowerin year. However, produced inthesecond family community. the characteristics by plants aredetermined studiedtowhatex communities. Wefurther focal to associated community influencesthenutritional weexamin field experiment Ina biodiversity This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedabundance ofarthropods will notaffectthe plant diversity willnegatively that thiswilllead toa decrease inconcentrationsof PAsinfocal plants impair resources), soil available and (for space We hypothesised: an (i)that increase inplant diversity willresult inincreased competition inthisplantspecies(Hol size have shownthat there isa positive relationship between the concentration ofPAsand plant ofhe species than70recorded fauna ofmore Naberhaus (e.g. for theirowndefence Article J. vulgaris Asteraceae Jacobaea vulgaris Jacobaea produces pyrrolizidine alkaloids(P pyrrolizidine produces . In the first year,a rosetteofleav In first the . Jacobaea vulgaris Jacobaea et al affect the number of affect thenumberof isabiennialorshort-lived . 2003; Schaffner . 2003;Schaffner plants that were planted plantsthatwereplanted quality and abovegroundquality and et al ht yellow inflorescences (Kostenko & Bezemer & yellow (Kostenko inflorescences ht . 2004). . 2004). the rosette is too small (Harper & 1957; istoosmall(Harper Wood therosette on reproductive focal plants. The reproductive reproductive focalplants.The on reproductive of the host plant and of the neighbouring plant host plantandoftheneighbouring of the tent the arthropod communities onthesefocal tent the arthropod ed how the diversity of theneighbouring thediversity plant of ed how rbivores (Harper & Wood 1957). Severalstudies & 1957). Wood (Harper rbivores c to a wide range of generalist insects, generalist of c toawiderange plant survival, development and growth, survival,development plant ed in Boppre 2011; Macel 2011). Specialist in Boppre 2011). 2011;Macel ed g may be delayed to later years when the years when may tolater g delayed be Jacobaea vulgaris e flowering stems can be morethan1m be stemscan e flowering arthropods on vegetative focalplantsbut arthropods onvegetative es is formed and flowering stems are are stems flowering and formed es is et al As), a well-studied group ofnitrogen- As), awell-studied . 2003; Kostenko et al. 2013). et al.2013). . 2003;Kostenko
perennial monocarpic plantofthe monocarpic perennial in the experimental plant arthropod communities harbours a rich insect arichinsect harbours . (ii)Anincrease in in the chemistry the focal of associated ofarthropods abundance diversity onthe We onthefocalplants. the abundanceofarthropods linkingcausal pathways thedi of alternative equa used astructural community. we Finally, ofeachplant characteristics of anumber recorded also We plants. reproductive and vegetative design determinedthe plants inafixed and plots withoutsurrounding pl (“bare vegetation maintained plant communities at nine one, species two,fourand diversity and levels, had weset-up In totestthesehypotheses, order plant community andthiscandirectly hi communities. in Vegetativeexperimental plants, diversitycommunity ofthat thereforehighly willbe they apparent inall toinsects mostoftheplantsin plants aretallerthan This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted tilledwitharotavator. each plotandthesoilwas In observation). Se (TM Bezemer personal intherestoration 1995. Theplantdiversity site was 180 Therestoration land. former arable within thenaturerest of25m×50 an area in thesummerof2008,70plots(3×3m),separate ofthefieldex A detaileddescription Experimental design methods and Materials Article
J. vulgaris periment ispresentedinKostenko plants. plants. nder insects from finding their host plant. fromfinding insects theirhost nder the surrounding community independent ofthe thesurrounding community independent ptember 2008, the vegetation was removedfrom 2008,thevegetation was ptember grassland is around 12-15 species per 3×m grassland isaround12-15species growth and primary and secondary chemistry of growth andprimary andsecondary a field experiment withplotsin whichwe experiment a field versity of the neighbouring plant community plant versityof theneighbouring to oration site Mossel, (Ede, The Netherlands) on siteMossel,(Ede,TheNetherlands) oration tion model to assess the strength and direction modeltoassessthestrength anddirection tion ots”). In each plot, we planted focal In plot,weplanted each ots”). contrast, are concealed by the neighbouring the neighbouring by concealed contrast,are ha and agricultural practices were ceased in ceased were practices andagricultural ha Plots weresownwithasingle plantspecies d by paths (1 m wide) were established in established were d by (1 m wide) paths hypothesized that (iii) the effect of plant hypothesized ofplant that(iii)theeffect to the focal plants is mediated by changes changes mediated by to thefocalplantsis
et al . (2012). Briefly, . (2012).Briefly, J. vulgaris 2
been weeded regularly, 25 25 regularly, been weeded sownplantcommunities In 2009,whenthe August site wasfenced. not mown.Toexclude plotswere experimental growing the season, the mownduring regularly betweenplotswere years 2009–2011.Paths untiltheendofth April) growing (late season sown species composition was maintained by enable comparing comparing enable andserved all vegetation, of free were kept were present communities. inmixed Four ofthese plotsinitiallya single sownwith species becau theexperiment from plots) wereexcluded monocultures of mixture) was replicated twice using a complete Informaticommunities, TableS1inSupporting mixtu 9-species andthree 4-species, eleven species L. millefolium Lotus corniculatus area (grasses: co thatnaturally grassland species with mixtures (monocultures) or This article is protected by copyright. All rights reserved. copyright. This articleis protectedby consists design experimental notincludedinthean plotswere The otherfour
Accepted Article vulgare , Tanacetum J. vulgaris L. Anthoxanthum odoratum , Hypochaeris radicata , Hypochaeris
T. arvense, T.maritimum was not sown. There were twelve di twelve sown.Therewere wasnot J. vulgaris J. vulgaris L. , Trifolium arvense, Trifolium L. , Tripleurospermum maritimum J. vulgaris of 66 plots. Initial of66plots. sowing 4000seedsperm was density performance inplotswithand performance of 2, 4, or 9 species randomly chosen from apoolof12local from chosen randomly of 2,4,or9species -occur (in high abundance) with abundance) (inhigh -occur seedlingswith at least twofully leaves developed were L. L., , Leucanthemum vulgare , Leucanthemum L. , Agrostis capillaris Agrostis , Trifoliumrepens res (12 + 9 + 11 + 3 = 35 different plant = 35different res (12+9113 A. capillaris as “no surrounding vege as “no hand-weeding from the beginning ofthe thebeginning from hand-weeding e growing season(late randomized design (35 × 2 = 70plots).The design (35×2 randomized on). Each plant community (monoculture or community on). Eachplant alyses of the experiment sothatthefinal oftheexperiment alyses large vertebrate herbivores the experimental experimental the herbivores vertebrate large se of poor establishment, butthesespecies ofpoorestablishment, se had establishedandthe fferent monocultures, nine2-species, monocultures, fferent and
(L.) W.D.J. Koch). The focal Koch).Thefocal (L.) W.D.J. without surrounding vegetation. A. odoratum L., other forbs: L. , Festuca rubra rubra , Festuca Lamk. J. vulgaris tation” treatment to tation” treatment August) throughout the August) , Plantagolanceolata (intotal4×2=8 four bare plotshad four bare inthestudied Achillea L., legumes: 2 . The was determined. At the end of August, plant ofAugust, At theend was determined. for48 was oven-dried plant remaining each of immediately at-20°C, frozen rosette andflower each leaffrom expanded in and placed clipped-off stems)were flowering plant Theaboveground plants couldbecollected. and 2) the 66plotslessthanfour(onaverage distributedamong notevenly was and flowering J. vulgaris and to collectfourreproductive reco focalplantswere 1324 (outof1650planted) years after 2011,two InAugust sampling Focal plantandcommunity W metalhalidelamps W week. Na timesper watered three inagreenhouse weeks grown forthree were into seedlingfilled trays withsterilized potting ger site.After vicinity oftheexperimental The without surrounding nootherplants thanthe25focal vegetation, community ofthesurrounding to testtheeffects m. Theresidentplantcommunity the surrounding 25 gridof 5 planted inaregular This article is protected by copyright. All rights reserved. copyright. This articleis protectedby
Accepted Article J. vulgaris J. vulgaris plants produced flowering stems(reproduc flowering plantsproduced plants × 66 plots = 1650 focal plants). plots=1650focalplants). plants ×66 rosettes were grown from seeds collectedgrown fromseeds were rosettesgrowing fromplants inthe direct (1 lampper1.5m × freeze-dried, weighed and ground for chemical analysis. The analysis. chemical ground and for weighed freeze-dried, 5 plants in the central 1.2 5plantsinthecentral four vegetative plants in each plot. However, only 424 ofthe only 424 ineachplot.However, plants four vegetative J. vulgaris tural daylight in the greenhouse was supplemented by 400 by wassupplemented inthegreenhouse tural daylight 2 ). mination, individual seedlings were transplanted mination, individualseedlings transplanted were rosettes had been planted in the field, atotalof hadbeenplantedinthe rosettes ing plant was removed with a razor blade, razor wasremoved witha plant ing (21/16 °Cday/night, 16hphotoperiod)and (21/16 community measurements were made in each madeineach were community measurements in 18 other plots (out of 66) no reproductive otherplots(outof66) in 18 h at 70°C, andtotalshootplantdry weight h compost. Before planting the field, in plants a labelled paper bag. The fifth youngest fifth fully bag. The labelled paper a on the establishment of the seedlings. In theestablishmentof theseedlings. plots on the plots (see Results). Therefore, in17of plots(seeResults).Therefore, the vered intheexperiment parts (rosettes of leaves or rosetteswith leavesor parts (rosettesof J. vulgaris The distance between theplantswas0.3 The distancebetween tive stage) two years aftertransplanting tive stage)two ×
1.2 m square of each plot(intotal 1.2msquareof plants was not removedinorder plantswasnot J. vulgaris al plots. We intended plots.We al were present. werepresent. arthropods per plant) and data from the four coll datafrom the four and arthropods perplant) (1 low relatively was date each at collected parasitoids).Th and (predators carnivores major groupsofarthropods: specializa degree of and the strategy feeding generalistherbivore, herbivore, po predator, we arthropod species S4).All level (seeTable Eppendorf 70% ethanolinlabelled atalldiversity leve amount oftimeateachplant all inspected collector over thefield.Each evenly distributed collectors by three anaspirator using collected were observed onaplant 10:00 inspectedbetween carefully plants were o four collected on plant were theirdevel Arthropods atallstages of Arthropod abundance analysis. chemical per plotandusedfor experimental each collected from were diameter cm and2.5 depth of15cm cores soil sampling, plant after week One each plot. height. a1.5-m Theheight from was released disc method(Stewart ofthevege The height community canoverlap. plot.Thetotalpercenta transect withineach percentage cover of plant species was recorded in two 1m two in recorded was species ofplant cover percentage plot, toestimate the structural complexity the community. of For each community, plant the This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article et al et . 2001). The disc weighed 200 g, had a diameter of 300mm,and of a diameter 200g, had . 2001).Thediscweighed specialist herbivores of specialist herbivores ccasions from May to August 2011. During each collection, all to August collection, May each 2011.During ccasions from plot at five random positions.Thesoilsamples plotatfiverandom werepooled tubes. Most arthropods wereid tubes. Mostarthropods opment (eggs, immature and adults) on each immatureandadults)on opment (eggs, e number of arthropods on individual plants ofarthropodsonindividual e number 1324 plants, spending an 1324 plants,spending st ge cover can exceed 100% because plantsina 100%because exceed can gecover llinator, detritivore ontheir based and omnivore) collection –0.4;2 collection tion. We focused our further analyses on three onthree analyses focusedourfurther tion. We was measured at 10 random locations within at 10randomlocations measured was re assigned to feeding group(specialist re assigned tofeeding am and 16:00 pm and all arthropods that were thatwere and16:00pmallarthropods am tation was measured using the vertical drop vertical drop using wasmeasured the tation ls. All arthropods were stored individually stored in Allarthropodswere ls. ections were therefore pooled for each plant. pooledforeach weretherefore ections J. vulgaris
2 quadrantsalongadiagonal nd , generalist herbivores, and generalist herbivores, and , –2;3 entified to speciesorfamily entified approximately equal approximately rd – 2; and4 J. vulgaris th –0.1
P according to Olsen Olsen to according P determined accordin Cwas organic percentage measur pHwas Scientific). (Thermo analyser insoilsamplesInc, C:N-ratio wa USA).The colorimetrically inthe CaCl content (NH N software. Mineral described inCheng Sepa Milford,MA,USA). (Waters, coupledtoaWate system chromatographic wasinjected solutionand10µl hydroxide filtrate wa oftheextracted filtration, 25µl solution containing heliotrine(1µg·ml groundplantmate freeze-dried of In 5mg brief, mass spectrometry following (LC-MS/MS) sampleswas and root PA analysis ofleaf Flash EA1112CNanalys a using were determined Leaf analyses. chemical the to were subjected intota resulting available reproductive plants vulgaris selected randomly we analyses, For chemical Chemical analyses S3). fashion (Fig. in aggregated plot withineach density) (independent oftheir arthropods of alsocalculatedoccurrences We This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article plants per each plot. However,insomepl plantspereach et al et al. et al. . (2011) and Appendix S2. Datawe (2011) andAppendix . (1954) and measured at 720 nm (Table 1). 720nm(Table 1). at (1954) andmeasured 2 extraction using a Traacs 800 au extractionusing 800 aTraacs 4 + andNO ration spectrometric and mass ofthe PAswas detection as -1 ) as internal standard. After centrifugation and standard.After ) asinternal carried out using liquid chromatography-tandem out using liquidchromatography-tandem carried in a Waters Acquity ultra-performance ultra-performance Acquity aWaters in s diluted with 975 µl of10mMammonium with975µl s diluted rs Quattro Premierta rs Quattro the procedure outlined in Kostenko outlinedinKostenko theprocedure 3 − s measured on a FlashEA 1112 SeriesNCsoil FlashEA 1112 s measuredona l 259 vegetative and 157 flowering plants that flowering plants l 259vegetativeand157 ) insoilsamples was determined ed in 2:5dry The ed in soil :watersuspensions. as the proportion of plants with arthropods proportionofplantswith asthe carbon (C) and nitrogen (N) concentrations concentrations (N) (C)andnitrogen carbon four vegetative and f g to NelsonandSommers g to to take into account that some arthropods occur thatsomearthropodsoccur to takeintoaccount rial was extracted with 0.5 ml 2%formicacid with0.5 wasextracted rial ots there were less than fourvegetative or er (Interscience, Breda, The Netherlands). Netherlands). The Breda, er (Interscience,
re processed using Masslynx 4.1 Masslynx using re processed toanalyser (TechniCon Systems (TechniCon toanalyser ndem mass spectrometer ndem mass our reproductive focal our reproductive (1982) and available andavailable (1982) et al . (2013). J. affected the abundance of arthropodsonvege of abundance the affected Fig. whetherthenum presented in S3.Totest linear general usingcommunity neighbouring a pe ofplantswitharthropods whether theproportion examined also We among plots. the plants offlowering distribution uneven because of Themodelsforthevegetative our experiment. in treatment wasmanipulated as it factor asfixed wasincluded diversity Plant eachplot. in plot identity to includedasrandomfactor was analyses In these analysis. the in diversity ofplant continuity the incorporate to factor fixed withplantdiversit models used mixed-effects abun ofplantdiversity onthearthropod the effect Proporti transformed. square-root of To fulfiltherequirements Data analyses This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Nconcentrations and between PA correlation chemistrygeneralconcentrations)(N and PA models linear were used (Table S7).The of proportionlegumes, as continu 1-9species) and species (0-9 diversity the vegetation and soilcharacteristics were analysed usinggeneral linear models withplant presente are data the of subset ofthe analyses using the analyses all repeated andthen plants, ofthearthr analyses we firstperformed thefoca asubsetof moment correlation.Asonly Article grasses, or other forbs in thevegetation on forbsin grasses, orother normality and homogeneity of variances data were log- or were log-or data variances of normality andhomogeneity ons data were arcsine square arcsine ons datawere opod abundance on the full onthe opod abundance d in Table S5. The effects of plant diversity on of Table S5.Theeffects d in product- a Pearson’s plantsweused tative plantsinthecommunity reproductive of ber y (0-9 species and 1-9 species) ascontinuous y species) speciesand1-9 (0-9 was tested using aPe was incorporate thatmultiple and reproductive plants reproductiveplants and smaller subset of the data. The resultsofthe The smaller subsetofthedata. model. The results of these analyses are are analyses Theresultsofthese model. l plants wassubjected l ous log-linear fixed factor. To test the effects effects the Totest factor. fixed ous log-linear dance, focal plant growth andchemistry, we plant dance, focal r plot was affected by the diversity of the diversity of the by r plotwasaffected
-root transformed. To examine Toexamine -root transformed. dataset including all arson’s product-moment J. vulgaris were run separately runseparately were to chemical analyses analyses to chemical plantsweresampled biomassand were chemically analysed. All variables used in the SEM were observed variables. To variables. wereobserved SEM the usedin variables All analysed. chemically were a excluded which bareplotswere plantswere andreproductive for thevegetative continuity the continuous factortoincorporate Plantdivers apparency. proxy thecommunity of competition withsurrounding a plantsforlight neighbouring ofthe vege The characteristics weusedplantshoot focal plantsinourmodels focal pathways linking and directionof strength modelling usedstructuralequation We (SEM Structural equation modelling Core Team2014). Rsta using analysed Datawere correlation. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedas herbivores, such tofocal associated fauna The arthropod Arthropod responses Results information abouttheSEMprocedur g package inR.Allfinalmodelsprovided the univariate analyses. Structural modelling equation carried was outusingthe improve the stabilise normalityand variances we data transformed same the as inthe in way Article J. vulgaris plants in biodiversity plots represented plantsinbiodiversity plotsrepresented Aphis jacobaeae Aphis
nd to develop thesemodelsweus nd todevelop Schrank (Hemiptera: Aphididae), (Hemiptera: Schrank e is presented in Appendix S3. inAppendix e ispresented J. vulgaris ood fit to the data (Table S3). Additional S3).Additional ood fittothedata(Table plant diversity and arthropod abundance onthe abundance and arthropod plantdiversity tistical language, version 3.0.1 (R Development language, 3.0.1(R version tistical tation included plant cover as a proxy as a of includedplantcover tation ) procedures (Grace 2006) to explore the explore to 2006) (Grace ) procedures biomass, nitrogen and biomass,nitrogen nd space, and height of the vegetation asa ofthe vegetation space, andheight nd of plant diversity Themodels intheanalysis. of run separately. We only examined modelsin examined only We separately. run ity (1-9 plant species) was included as fixed asfixed included was species) plant ity(1-9 plants was dominated by specialist specialist by was dominated plants in the Fig. 1. As characteristics of the ofthe 1.Ascharacteristics Fig. the in
ed the subset ofplantsthat ed thesubset total PAconcentration. Tyria jacobaeae Tyria lavaan
L. L. (0-9 species: F (0-9 species: P F F species: (0-9 plotdecreased plantsper focal flowering F species: F community neighbouring (0-9species: ofthe diversity increasing arthropodsassociat ofcarnivorous abundance F species: diversity (0-9 abundance of generalist herbivores F with increasing plant bare diversity when pl herb ofspecialist abundance 0.0063, Fig. The 2). reproductive plants inthecommunity ( reproductive plants onvegetativeplants arthropods of The abundance associated toreproductive ofarthropods abundance F communitythe neighbouring (0-9species: vegetative on number ofarthropods thetotalnumberof for 87%of that accounted and (Lepidoptera: Arctiidae) This article is protected by copyright. All rights reserved. copyright. This articleis protectedby P F species;shoot: (0-9 plantbiomassofvegetative affect 1). diversity (Table Total plantand cover height ofthe vegetation increased significantly withincreasing plant Plant communityandfocal 1,60 1,64 = 0.39, Fig. 3) focal plants. The leaf N concen Theleaf focal plants. 3) = 0.39,Fig. Accepted shoot:F (0-9species; and reproductive = 0.19,Fig. 3) Article =2.80, =4.99, 1,60 P P =6.09, 1,64 = 0.029) with increasing plant diversity. Plant diversity did notsignificantly with increasingplantdiversity. did Plantdiversity = 0.029) = 0.099;specialists;1-9species:F =16.41, J. vulgaris J. vulgaris P = 0.017, Fig. 2). There was no effect of plant diversity onthe ofplantdiversity noeffect was = 0.017,Fig. There 2). 1,64 =1.08, P = plant characteristics Longitarsus jacobaeae survival (F 0.0001, Fig. 3) and C:N ratio increased (0-9 species:F (0-9 C:Nratioincreased and 0.0001, Fig. 3) on vegetative plants was not significantly affected by plant by plants wasnotsignificantly affected onvegetative P J. vulgaris = 0.30;1-9species:F P > 0.05 in all cases). all cases). > 0.05in 1,64 ots were excluded from the model (0-9 species: (0-9species: fromthemodel excluded ots were 1,64 =12.05, ed to vegetative plants decreased with ed tovegetativeplantsdecreased collected arthropods (Table S4). Thetotal (Table arthropods collected plants decreased with increasing diversity of diversity withincreasing plantsdecreased = 6.0,
tration of vegetative focal plants decreased plants decreased focal ofvegetative tration ivores on vegetative plants also decreased plantsalsodecreased onvegetative ivores 1,60 did not correlate with thenumberof didnotcorrelate Waterhouse (Coleoptera: Chrysomelidae) (Coleoptera: Waterhouse J. vulgaris =4.33, P P 1,64 1,46 1,64 = 0.017; 1-9 species: F = 0.017;1-9species: = 0.0009) and the number of = 0.0009)andthenumber
=1.16, =0.37, 1,60 =5.84, P =0.60, = 0.042, Fig. 2) whereas the 2)whereas = 0.042,Fig. plants( P P 1,64 P = 0.29;root:F = 0.55;root:F = 0.019; 1-9species: =5.05, P P > 0.05 inallcases). > = 0.44,Fig. The 2). P 1,60 = 0.028;1-9 =8.03, 1,64 1,46 1,64 =1.77, =1.31, = P = (accordingly) associated to the focal focal the to associated (accordingly) arthropods,specia ofcarnivorous the abundance The final SEMforvegetative e SEM: Directandindirect r =0.40, species: r=0.55, r=0.54, (0-9species: forbothvegetative withNconcentration correlated (F focal plants of vegetative reproductive focal plants ( ordi concentration diversity onthetotalPA notincludedinthe bareplotswere 4). When F (0-9species: plants focal and seneci jacobine- for diversity observed was reproductive: F concentration was statistically e (Fig. 4).The community of theneighbouring plan focal ofthe concentration PA the Overall, plants( reproductive vegetative and on fo of plantdiversity no effect toplotswith werelimited When theanalyses wa plants focal reproductive of leaves in Fig. 3) species: F species: 10.60, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article P = 0.0018, Fig. 3) with increasing plant diversity. The concentration ofN(0-9 concentration The plantdiversity. withincreasing = 0.0018,Fig. 3) P 1,46 < 0.0001) plants. plants. <0.0001) =3.38, 1,46 P =1.64, < 0.0001) and reproductive (0-9 species: r = 0.34, species:r (0-9 andreproductive < 0.0001) P = 0.072, Fig. 3) and C:N ratio (0-9species:F = 0.072,Fig. andC:Nratio 3) P P 1,64 ffects ofplantdiversity > 0.05 in all cases) except on senecionine-type PAs intheleaves onsenecionine-type except >0.05inallcases) 1,60 = 0.21, Fig. 4). However, a significant negative effect ofplant effect negative asignificant = 0.21,Fig. 4).However, =6.76, J. vulgaris not-significant (vegetative: F (vegetative: not-significant =5.35, cal plant biomass, leaf Ncon leaf biomass, plant cal P J. vulgaris > 0.05 in all cases). cases). >0.05inall P P = 0.012;F plantsexplained23%,6%a = 0.024). Total PA concentration positively= 0.024).TotalPAconcentration fferent types of PAs for both vegetative and bothvegetative types ofPAsfor fferent model there was nosignificant effect ofplant surrounding therewas vegetation(1-9species), ffect of plant diversity on the totalPA ofplantdiversityffect onthe ts tended todecrease withincreasing diversity plants (Fig. 5, Table S3). There was a direct adirect was plants(Fig. 5,TableS3).There onine-type PAs in the leaves of vegetative ofvegetative leaves the in PAs onine-type s not significantly affected by plantdiversity. not significantly affected s list herbivores and ge list herbivoresand
1,64 =12.34,
1,64 centration and C:Nratioforboth and centration =3.04, P = 0.0001 respectively, Fig. Fig. = 0.0001respectively, 1,46 nd 6% ofthevariation in nd 6% neralist herbivores neralist P P =3.34, < 0.0001; 1-9species: < = 0.086, P < 0.0001;1-9 P = 0.074, effects of plant diversity on the abundance of on theabundance ofplantdiversity effects of associated withPAconcentration abundance ofspecialist ofthe inturnreducesthebiomass community that cover ina plants(-0.076, inthefocal concentration 0.12, linksfromplantdi negative indirect there were than arthropod abundance variation incarnivorous 0.08, J. vulgaris inthe vegetation cover arthropods viaincreased surrounding community. Secondly, plant divers carnivorous of theabundance diversity enhanced also twoindirect diversity had plantdiversitynegative pathfrom totheabundan This article is protected by copyright. All rights reserved. copyright. This articleis protectedby herbivores through the indirectpositivepathway betweenplant herbivores positively associated was ofthe tothe focal biomass focal variationintheabunda 1%ofthe herbivores and arthr ofcarnivorous abundance The finalSEMforreproductive P not plant PAconcentrationwere Accepted =0.14). Article J. vulgaris P P = 0.030) through the increased percentage percentage the increased =0.030)through = 0.099and-0.09, focal plants. However, thei However, focalplants. plants by indirect pathways (Fig. plantsby pathways (Fig. indirect J. vulgaris was negativelyofgene andthat P = 0.053) were offsetting and less = 0.053)wereoffsetting andless shootbiomasswasnotsignificant (0.22, effects on the abundance of carnivorous arthropods. First, plant arthropods. carnivorous of abundance onthe effects opods, 36% of the variation in opods, 36%ofthe J.vulgaris significant (specialists: 0.026, (specialists: significant J. vulgaris ndirect pathways (thestrength ndirect pathways P = 0.034) through the increased percentage plant percentage increased through the =0.034) plants explained 47% of the variation inthe 47%ofthevariation plantsexplained diversity and the abundance of generalist abundance of andthe diversity herbivorous insects mediated by changes in by changes insectsmediated herbivorous versity to N concentration in focalplants(- in N concentration versity to focalplants(Fig. 5). ity decreased the abundance of ity decreased carnivorous the abundance community theshootbiomassof thatreduced nce of specialist herbivores associatedtothe herbivores ofspecialist nce arthropods by promoting the height ofthe height by arthropods promoting the ce of carnivorous arthropods (Fig. 5).Plant arthropods ofcarnivorous ce 5, Table S3). The abundance of generalist generalist of 5, TableS3).Theabundance plant cover in a community, andtoPA plant coverina the direct pathway (-0.40). Interestingly, (-0.40). the directpathway ralist herbivores was positively herbivoreswas ralist
J. vulgaris the abundance of generalist of theabundance important in explaining the important inexplaining P J. vulgaris =0.14;generalists: -0.019, However, the indirect theindirect However, of the indirect effect = effect indirect the of P plants.The = 0.063).The = plants.However, vegetative and Sn-typ almost allPAgroups Jb-type (i.e. isinfluencedby compounds inafocalplant the field,com Our study showsthat,in Discussion was notsignificantlyassociated significant (-0.13, thebiom lowering and cover inthecommunity path showing negatively thatplantdiversity and plantdiversityconnecting (F ourexperiment in measured characteristics associ butwasnotsignificantlyabundance arthr abundance ofcarnivorous This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted focal in concentrations fo explanations possible are several There ofplantdiversity plant) effects chemistry andsubseque diversity onplantdefence diversitychemistry onthe focalof the plants. vegetative on arthropod abundances community.neighbouring SEMrev However, plant also d focalplants arthropods foundonthese intheplotsw plant species)andhighest Article J. vulgaris P = 0.037). Chemistry of reproductive focal Chemistryreproductive =0.037). of plants were lowest in the plots w plantswerelowestintheplots J. vulgaris J. PA concentration inreproductiv concentration PA opods was strongly positively linked to the specialist herbivore positivelyopods wasstrongly linkedtothe on aboveground arthropods on focal plants. onfocalplants. arthropods aboveground on with arthropod abundances (Fig.5). (Fig.5). abundances with arthropod plants. Increasinggenerally leadstoan plants. plantdiversity J. vulgaris ithout surrounding vegetation. The abundance of ithout surroundingabundance The vegetation. r the observed diversity effects onPA effects diversity r theobserved position andconcentrati ated to any focal plant or community community focal plantor any ated to the neighbouring plant community. The levels of the neighbouring Thelevels plantcommunity. affects PA concentration by promoting PAconcentrationby plant affects ecreased with increasing diversity of the of diversity withincreasing ecreased e PAs) andthetotal e PAs) ig. 5). There were several indirect pathways pathways indirect several were There 5). ig. Below, we first discuss the effects of plant ofplant wefirstdiscusstheeffects Below, ass ofthe focal plants, was statistically were not mediated by the effects of plant ofplant effects the werenotmediatedby ntly the direct andi ntly thedirect ealed that the effects of plantdiversity on of theeffects ealed that ith the highest species diversity(nine
plants (N or PAconcentration) or plants(N e plants, but only the negative butonly thenegative e plants, PA concentration of PA concentration on of secondary plant on ofsecondary ndirect (via the focal focal the (via ndirect vulgaris studieshave Several resources. for available forbs, fo communities withhigh abundanceof reproduc vegetative and the affected negatively community neighbouring vulgaris ofope thenumber andthusreducing effects) plant community (whi surrounding cover ofthe growthdiversity the ofvegetative indirectly suppressed (McEcoy plants focal ofthe growth reduced to can lead vulgaris et al. light, aswe and competition forspace areusuall communities More productiveplant inGross oftheplantcommunity productivity intheoverall (reviewed increase This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted 5). Fig. (seealso size plant on effects diversity by fully mediated not are chemistry defence onplant effects diversity the of plant diversityPA concentration onthe plants remainedS6) suggesting infocal (Table that wa ofplantsize theeffect Importantly, when in smaller plants, which, inturn,led toth speculateplant that diversity increased led toincreased competition for withshootbiomass highly positively correlated shoot biomassinbothreproductiveandvege a positive SEM revealed 2003). Alsoinourstudy Article 2011; Lorentzen 2011; plants ispositively related tothe rootbiomass (Hol rosette development (McEcoy andincreasedcomp isapoorcompetitor et al tive) to produce PAs. tive) toproduce . 2008;Eisenhauer ll assoilnutrientdepletion(Spehn et al e observed decrease in PA concentrations. inPAconcentrations. decrease e observed . 1993). Inthat forbsinthe addition,wefound . 1993). tative plants.Astherootbiomassof tative n spaces on the ground that are essentialfor groundare onthe that n spaces s removed fromthe models,significant a effect shown thattotalshootPAconcentrationof shown J. vulgaris cal plants sufferedfro cal y denser, which can lead to increased y whichcanlead toincreased denser, et al et ch we assume is a proxy for competitive for we assumeisaproxy ch in the field (r = 0.96; data not shown)we not in thefield(r=0.96;data etition inmorediverseplantcommunities growth and the ability of focal plants(both focal growth andtheability of path between the total path betweenthe . 2009). The focal plantinourstudy,. 2009).Thefocal J. vulgaris et al is a forb species and possibly, isaforbspeciesand in
et al. . 1993).SEMshowsthatplant 2003; Schaffner 2003;Schaffner plantsbypromoting the m increased competition competition m increased J. vulgaris PA concentration and PA concentration et al. 2000;Oelmann et al J. vulgaris , resulting et al . 2014). . J. J. J. is plant, and in our study foliar N concentration in Nconcentration plant, andinourstudy foliar the soil. Hence,concentration leaf may N be be whil endoftheseason, atthe once only measured Itcommunities (TableS7). isimportanttonot (reproduc plants) ornotaffected (vegetative weakly shown)andwas not (data Ncontent mineral soil with correlated was not plantcommunities(Tab abundance inmorediverse communities asinmonospecific communities, plant diverse high as high in wastwice and diversity plant increasing with increased soil the though wasnotstatistically therelationship signif in plant diversity could therefore leadN availability toreduced However, even inthe soil. resources more effectivelythan simple communities (e.g. Oelmann Mattson 1992).Someauthorshave the availabilityresources of inthe soil(Bryant production ofplantde thatthe Theory predicts This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Wäschke plantsviathe compounds infocal defence Previous studiessuggested thatplant diversity at therosettestage isstronger concentration plants,itsuggests was only forvegetative true didnotinclude (thepathway that concentration diversity andPAco plant pathway connecting both vegetativeandreproductive diversity.Foliar Nconcentration was positively correlatedwith total PAconcentration for Article et al. 2015). For example, Mraja example, 2015).For tive plants) by the abundance of legumes in the neighbouring oflegumes in abundance the tive plants)by plants. However, SEM revealed a significant indirect indirect a significant revealed SEM However, plants. argued that diverse plant communities may plantcommunities argued thatdiverse uselimiting
effects of diversity on herbivory (Mraja ofdiversity onherbivory effects and colleagues (2011) reported that increased thatincreased reported colleagues (2011) and when the plant has a low shoot to rootratio. the planthasalowshoot when e, though, that N availability in thesoilwas availabilitye, though, thatN ncentration via changes inleaf nitrogen that the correlation between plant NandPA between thecorrelation that fence compounds can be (partly) explained by explained be(partly) compoundscan fence can also influence the concentration ofplant concentration also influencethe can possibly due to an increase inlegume due toanincrease possibly al et tter indicator ofnitrogen availability tothe focal plants decreased with increasing plant plant increasing with decreased plants focal plant size) only for vegetative plants.Asthis onlyplant size) forvegetative icant, in our study, the mineral N content in mineralNcontent inourstudy, the icant, e plants had been growing for two years in two for growing e plantshadbeen le 1, Fig. S4). The total PA concentration le 1,Fig. S4).Thetotal . 1983;Coley
et al et al . 1985;Herms& . 2011). An increase Anincrease . 2011). et al. 2011; differen not mediatedby the (Hol in demonstrated thatPAproduction diversity plots. experimental induction) relatedtodifference (defence weredirectly concentrations PA in differences the that possible is it Therefore, to the prediction ofthe accordance in is finding This diversity. plant increasing with decreased herbivores specialized as previousstudy wasoppositetothe effect to thenumberofspecialised focal plants. However, similarly tothe previous In study, wedidnot experiment. thepresent damage atincreasin withherbivore correlated in compound aninduceddefence ofcatalpol, concentration This article is protected by copyright. All rights reserved. copyright. This articleis protectedby on effect have astrong vulgaris forchanges beresponsible also may effects S7), plant functionalgroups(Table communi neighbouring ofthe other characteristics va genetic we assumethatthe origin plants asallfocal However, al basis. ThePAcompositionin ofpl intheexpression variation Intraspecific
Accepted. 1993;Macel Article et al . For example, inthepreviousstudies itha . For example, . 2004). Thus, in our system the diversity effects on plant chemistry are mostlikely onplantchemistry. 2004).Thus,inoursystem are thediversityeffects et al . 2004). We did not measure genetic didnotmeasure . 2004).We J. vulgaris J. specialist-generalist dilemma herbivores colonizing herbivores ces in herbivore abundances. abundances. ces inherbivore However, itisimportanttonot riation among individualplants J. vulgaris s in herbivore pressure onthe pressure s inherbivore ated from seeds collected from one from collected seeds from ated biomass allelopathic effects, orin allelopathiceffects, J. vulgaris plants ispartiallygenetically (Vrieling determined
and PA concentration via changes in the the in changes via concentration and PA in plant growth and PA concentrations of growthand PAconcentrations in plant both the PA concentration and the numberof and both thePAconcentration estimate theamountofhe ant defence compounds can also have a genetic havea also compoundscan ant defence g plant diversity in a grassland biodiversityggrassland diversity plant in a is not induced in response to shootherbivory isnotinducedinresponseto study PAconcentration waspositivelyrelated ty, such as the identity and diversity of and identityty, suchasthe s been shown that other plant species can speciescan s beenshownthatother plant J. vulgaris J. vulgaris hypothesis (van der Meijden 1996). der hypothesis (van
variation among the focalplants. among the variation Plantago lanceolata e, that previous experiments experiments e, thatprevious terspecific plant-soil feedback plant-soil terspecific focal plants in the different plantsinthedifferent focal was relatively low.Finally, rosettes. Although thediversity J. vulgaris J. vulgaris rbivore damageonthe rbivore , positively population, J. et et (even in the short term) can pers can (even intheshortterm) variation intraspecific beenshownthat has also nutrientcyclin dynamics, herbivory, disease a wi for consequences haveimportant scales can 2011; Wäschke compoundswere samedefence even whenthe inourstudy butal from thoseobserved differed Wäschke far (Broz studiesso few by addressed compounds hasbeen questionhowplant the aware, as weare As far investigation. further thisneeds However, Heil & 2010). Karban ofdefens expression the inducing focal plantby plan Emission ofvolatilecompoundsby neighbouring deVoorde composition ofthesoilmicrobial community (Van This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedthe rosette stage (Kostenko experimentalyear plotsduring after the transplantation first when all theabundance reported we Ina previous study chemistry. defence tounde metabolites arenecessary invol pathways ofbiosynthetic an understanding defe ofplant plasticity the examine that studies generation that isaninterestingnew question offocal inthePAprofiles differences Article et al . 2015). The strength and direction ofpl anddirection strength . 2015).The et al . 2015). Intraspecific variation inplan Intraspecific variation . 2015). et al . 2012). In on arthropods study,. 2012). collected thecurrent we ist in new generations (Hennion generations ist innew rstand and predict theeffects rstand andpredict J. vulgaris g and decomposition (Crawley 1997). Recently, it decomposition(Crawley1997). Recently, and g should be investigated in the future. Field should beinvestigated future.Field inthe examined (i.e. iridoid glycosides; Mraja (i.e.iridoidglycosides; examined nces inrelation toe of insects on the same focal plants inthe insectsonthesamefocal of diversity affects the levels of plant defence of plantdefence thelevels affects diversity so varied among the abovementioned studies in plant chemistry by triggered plant diversity ive chemicals in the focal plant (reviewed in (reviewed inthefocalplant ive chemicals de variety of ecosystem processes, such as such processes, ecosystem of variety de ved in the synthesis of particular defensive defensive ved inthesynthesis ofparticular plants inourstudy willbe inherited by the ant diversity effects in thosestudies in effects ant diversity ts may a theresistanceof alsoinfluence
t defence chemistry chemistry t defence et al et al of plantdiversity onplant . 2010; Mraja . 2010;Mraja et al nvironmental factors and factors nvironmental . 2011;Kos . 2016). Whether the Whether . 2016). J. vulgaris et al at smallspatial et al were stillin . 2011; . 2015). et al . the apparency of vegetative plantswasalso ofvegetative the apparency appare in withthedifferences along Finally, vulgaris focalplants.Howeve compared toreproductive ar themuchlowernumberof also explain flowering the than for insects apparent th during flowering plants inthoseplotswere lownum may duetothe been have growing intheba plants the vegetative focal inth experiment, year the first during of the to individual plants growing inthose communitie most diverse communities. Thissuggests diversity that plant associational provides resistance specialisedherb years fewer as wefoundinboth plots.Theresultsofthecurrent experimental pl focalplantswhen reproductive vegetative and This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted fromde parasitoids andpredators of levels andcomplexity volatile blends) ofplotswithdifferent ofdiversity levels notmeasured that here. was High possible thatthisnegative direct in contrasttothe“ thevegetative plantsrema on carnivorous arthropods direct negative path the plant characteristics, After correlations accounting among diversity, forpartial plant characteristics vegetation and neighbouring community. Article plants did not correlate with thenumber plantsdidnotcorrelate Associational susceptibilityhypothesis” Associational plant odours in more diverse plant communities might hinder plant odoursinmorediverse plantcommunities effect is a result of differenc is aresult effect tecting the host cues (Wäschke (Wäschke cues host the tecting ber of vegetative plants present in the bare plotsasmost plantspresentinthebare ber ofvegetative J. vulgaris thropods thatoverallwe thropods e second year very few arthropods were found on were arthropods year few very e second significantly affected by the diversity of diversity the by affected significantly ncy between reproductive and vegetative plants and reproductive ncy between re plots withoutneig re plots linking of totheabundance plantdiversity e second year, and because the rosettes are less rosettesare the year,because and e second study are in agreement with the previous study withthepreviousstudy study inagreement are r, the abundance of arthropods onvegetative ofarthropods the abundance r, ivorous insects on vegetative plantsinthe insectsonvegetative ivorous years inthe for two growing hadbeen ants s. However,results incontrast obtained to of the reproductive plants in acommunity. plants thereproductive of plants. The difference in apparency may inapparency plants.Thedifference ined significant intheSEmodels.Thisis
and the“ es in chemical profiles (e.g. profiles(e.g. es inchemical hbouring vegetation. hbouring This et al. re found on vegetative found re Enemies hypothesis” Enemies 2014). It 2014). isimportantto . It is J. number of arthropods present onthesepl present number ofarthropods tothevegeta associated carnivorous arthropods studies (Moreira mutualists herbivore roleofsuch honeydew. The al explained by the aphid-ant mutualism that isknown forthisspeciescombination (Vrieling aphid recorded predators ofthe Asmany abundance. betwee positivecorrelation a strong there was vulgaris e indirect was nodirector toindividualpl trophic levelinsectsassociated dise neededtofurther methods suchastrapsare of parasitoidsorothervisual notice thatthemethodusedinourstudy to This article is protected by copyright. All rights reserved. copyright. This articleis protectedby abundances of generalist herbivores increased not forspecialist herbivores that may use more attr more arelikely that predators and herbivores Schlinkert had highstating plants thatlarger affect positively focal plantswere In our Leather 2002). & inAwmack (reviewed host-plantsele including insect performance, Plant quality (primarysecondary and compounds) is
Accepted ArticleIn notc . 1991). herbivoresare the thiscase Aphis jacobaeae plantsdespitethehigh ofarthro number et al . 2015). Interestingly, thispathway. 2015). et al . 2016). In contrast, there were noa In were . 2016). contrast,there was the most abundant specialized he themostabundantspecialized was ffect of plant diversity on on ofplantdiversity ffect ly oriented predators. More predators. ly oriented er arthropod abundances (Castagneyrol (Castagneyrol abundances er arthropod ed by the plant size. This is in accordance with the theory Thisisinaccordance size. theplant ed by ants compared to reproductive plants. plants. toreproductive ants compared collect arthropods might ction, growth, survivorship and reproduction ction, growth, survivorshipand onsumed by the predator but are tended for butaretended by thepredator onsumed n specialist herbivore abundance and predator andpredator n specialistherbivoreabundance on the flowering plants were antsandthe plantswere the flowering on and of specialist herbivores decreased with decreased of specialist herbivores and ants. In agreement with our hypothesis there specific host related cues. Furthermore, the cues. specifichostrelated study, the arthropod abundances associated to associated arthropod abundances the study, tive plants, perhaps as a result ofthelow as aresult tive plants,perhaps ntangle the effects of plant diversity on higher onhigher ofplantdiversity effects the ntangle pods recorded on these plants. However, ontheseplants. recorded pods acted tolarger and extr have been largely overlooked inbiodiversity overlooked largely have been was more important for generalist moreimportantfor was arthropod abundance on reproductive on arthropod abundance extremely important in every aspect of importantinevery aspect extremely studies using other collection studies using other ssociations between herbivorousand ssociations between
rbivore, this relationship mayrbivore, thisrelationship be underestimate thenumber underestimate a apparent plantsand a apparent et al. et 2013; J. et et In summary, using a field experiment, wher experiment, Ina field using summary, arthropod relationship with reveal the Koricheva and therefore 2010) plant chemistryat one measured time pointmay notproperly season. Plantchemistry is knowntovary w whereasthearth season growing during the chemistry.It tomention that isimportant the chemistryfocal ofthe plants measured was once also were inourstudy on thearthropodabundances herbivores (Wäschke compounds in concentr the where practices, use land different onplantchemis complexity of vegetation effects ye forthisresult donothaveanexplanation We SEM analysis tothe andisincontrast focal ofthe concentration PA in increases This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted disentanglingplant quality of the role instructuringcommunities insect innatural settings. acommunity perspect from should beconsidered emphasi Ourstudy plants. focal the to associated inpl intraspecific variation diversit increasing with arthropods decreased compounds secondary ofthemajor concentration that arthropods aboveground of and theabundances nutritional qualityand sec thatthediversit weshow experimentally, Article Plantago lanceolata et al. et ondary chemistry of individual plantsgrowing thatcommunity individual ondary in chemistry of 2015). However, as shown by SEM the effects of plantdiversity by SEMtheeffects asshown 2015).However, ant defence chemistry did not ant defencechemistry L.) did not correlate with the abundances ofspecialist L.) withtheabundances didnotcorrelate specialist-generalist dilemma abundances of an entire growing season. season. growing of anentire abundances y of the neighbouring vegetation affects the affects neighbouringy vegetation ofthe J. vulgaris ith plant seasonal development (Barton & seasonal development ith plant ropods were collected four timesduring the collected were ropods e plant species diversity was manipulated wasmanipulated diversity species e plant y of the neighbouring community. However, the However, y community. neighbouring ofthe ation of iridoid glycosides (major defence defence ofiridoidglycosides (major ation t. It contrasts a previous study aprevious the examining It contrasts t. try and insect community in grasslands with communitytry in and insect zes that individual plant-insect interactions thatindividualplant-insectinteractions zes ive. Future studies shouldaimatfurther ive. of the focal plants and the abundance of plants andtheabundance ofthefocal not mediated by the changes in focal plant infocal changes notmediatedby the naturally colonise the focal plants.The colonisethefocal naturally rosettes. Thisisa surprizing result ofthe affect arthropod communities affect
(van der Meijden1996). (vander noconflictofinterest. declare This ispublication6188Inof theNetherlands of Netherlands Organization previous versionofth valuable commentsona vande andWim Irisanalysis forCN Chardon a EkeHengeveld Drok, Roel Wagenaar, Sylvia studyWe thank Saskia ontheir property. Grootemaat,Budak, Hanife Ciska Raaijmakers, (Planke grateful toNatuurmonumenten are We Acknowledgments revisions. draftofthemanus thefirst the dataandwrote andMCpe OK analyses, chemical performed Author's Contributions This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted (1976)Plantdefense guilds. D. J. Atsatt, P.R.& O'Dowd, herbivory, andplan root ofmilkweed:Competition, andsusceptibility A.(2004)Resistance Agrawal, References plantchemistryand insects). on effects 2016. Diversity http://mda.vliz.be/mda/direc Data depositedintheMarine Data accessibility Article t genetic t genetic variation. : OK and MB designed the experime the OKandMBdesigned : tlink.php?fid=VLIZ_00000241_1394710219 (Kostenko tlink.php?fid=VLIZ_00000241_1394710219 Data Archive repository: repository: DataArchive Scientific research (NWO, VIDI grant no.864.07.009toM.B.). VIDI (NWO, Scientific research Ecology, cript, and MB contributed substantially andMBcript, to contributed rformed arthropod identification, OK analysed OKanalysed arthropodidentification, rformed r Putten and three anonymous reviewers for reviewers anonymous andthree r Putten stitute of Ecology (NIOO-KNAW). Authors (NIOO-KNAW). stitute ofEcology is manuscript. This work was funded by the fundedby manuscript. Thisworkwas is n Wambuis) forpermissiontoperformthis Wambuis) n nd Roeland Cortoisfor nd
85, 2118-2133.
Science, nt and collected thedata,PM nt andcollected
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86, 177- Appendix S4. Appendix S3. Appendix S2 Appendix S1. Additional supporting may information found be Supporting Information elder herbivore. A.&White, Whitham,J. T.G.(2000)Associ parasitoid orientation? odourenvironments:How Habitats ascomplex Hancock,C.,Hilker, K., N.,Hardge, Wäschke, landscape? complexity affecthostplantchemistry, and C., Hilker N.,Hancock, Wäschke, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedmissing files)shouldbea support issuesar Technical edited ortypeset. may bere-org authors. Suchmaterials by the this andreaders, toourauthors As aservice Appendix S5. Article Oecologia, . Additional information onpyrrolizidine alkaloids Additional data analyses Additional dataanalyses Arthropodcommunity responses SEMprocedure sownintheexperimental plots Speciesmixtures Ecology
Plos One, 179, , ddressed to the authors. ddressed totheauthors. 81 281-292. , 1795-1803.
9 , M., Obermaier, E. & Meiners, T. (2015) Does vegetation Doesvegetation T.(2015) E.&, M.,Obermaier, Meiners, , e85152. , e85152. journal providessupportin journal thus multitrophic interactions, in a human-altered multitrophic interactions,inahuman-altered thus anized for online delivery, but are not copy- are not but delivery, foronline anized ising from supporting information (otherthan fromsupporting information ising ational susceptibility of cottonwood to a box to abox ational susceptibility ofcottonwood M., Obermaier, E. & Meiners, T.(2014) E.& Obermaier, Meiners, M., does plant does herbivoreand diversity affect in the online version of this article: theonlineversionofthis in
g supplied information 16 . 1. . 51 .4 68±03 37 .6 1. . 40 0.09 4.04 ± 114.5 0.15 ± 1.66 0.07 3.77 118.7 ± 3.71 ± 3.97 ± 114.7 5.0 ± 0.06 1.02 0.39 ± 3.82 ± 115.4 0.94 1.86 ± 8.3 ± 16.8 3.95 ± ± 1.9 0.23 0.47 0.08 2.15 0.04 ± ± 16.4 2.5 0.07 5.13 ± ± 16.7 0.10 3.97 ± 117.2 0.05 0.8 ± 16.6 ± 5.13 ± 0.01 0.37 5.17 ± 1.91 ± 2.1 0.01 13.3 ± 5.14 ± 0.4 0.13 ± 166 ± 16.7 0.4 11.2 ± 8.2 0 ± 0.02 ± ± 5.10 ± 160 8.8 6.3 1.4 included ± 152 0 10.2 ± 7.0 Bare plots ± ± 132 7.0 0 0 9 4 2 1 0 plant Total diversity Plant excluded excluded Bare plots diversity of the neighbouring plantcommunity diversity oftheneighbouring changes inthe communitycharacteristics focal orquality of Fig. 1. significant not is effect the that indicates asterisks ** themodel.Aste from included orexcluded model w linear general based ona or keptwithoutvegeta with 1,2,4or9species Table 1. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby as total per plotiscalculated value average The SE). ± between-plot plot per values on average based shown (calculated focal reproductive thevegetative and on carnivorous arthropods abundan specialist herbivore Fig. 2. arrows grey light the by arerepresented diversity ofcommunity effects indirect the arrow; community onarth of diversity direct effect wasincluded treatment andthatthisvariable vulgaris cover (%) F Accepted ArticleP F 1,60 1,64 < 0.01; the brackets indicatemarg <0.01;thebrackets Effect of diversity of theneighbourin ofdiversity Effect =95* F =9.52** =3.5* F 34.25*** = Initial conceptualmodel describing the direct potential (mediated and indirect by the plants. The hexagon around the“Plantdivers plants.Thehexagon around Vegetation and soil characteristics (mean ± SE (mean characteristics soil and Vegetation height (cm) (cm) height Vegetation 1,60 1,64 =1.1* F 11.31** = F 43.11*** = ce, generalist herbivoreabunda generalist ce, number of arthropod individuals in number ofarthropod pH C:N pH ratio ith plant diversity as fixed log-linear factor and bare plots andbare log-linear factor fixed ith plantdiversityas 1,60 1,64 =(.8 F F =(3.88) =3.15 inally significant effect at effect significant inally risks indicatesignif ropod abundance is represented by dark grey by dark isrepresented abundance ropod g community on total number of arthropods, g of community ontotalnumber as fixed continuous factor in the models.The continuous factorinthe as fixed on arthropod abundance associated to focal tofocal associated arthropod abundance on tion (0). Asterisks indicate significant effects effects (0).Asterisksindicatesignificant tion 1,64 1,60 =13 F =1.36 =05 F =0.58 ity” variable indicates themanipulated indicates ity” variable ) of experimental plotsthatweresown ) ofexperimental
nce, and the abundance of and theabundance nce, J. vulgaris icant effect at*** icant effect (mg·kg (NH mineral Soil N aplotdivided number by the 1,64 1,60 J. vulgaris P =22 F =2.29 =18 F =1.82 4 < 0.06; the absence of the absence <0.06; + +NO -1 ) plants) effects of effects plants) 3 − ) plants. Means are Means are plants. P (mg·kg 1,64 1,60 P =09 F =0.92 =04 F =0.44 <0.001; -1 ) J. (%) matter Organic 1,60 1,64 =0.85 =1.91 0.01; * tothe arrows that areshownnext retained model. inthe Arrowwidthsare final dashedlin The respectively. effects, vulgaris a arthropod and characteristics characteristics relationships among the Fig. 5. sp relationship withplantdiversity (0-9 focal seneciphylline-typePAs (mg·g (Sp) eruc (Jb), ofjacobine-type the concentration Fig. 4. model. onthemixed-effects species) based with plantdiversity (0-9 vulgaris andC:Nratio biomass, leafNconcentration Fig. 3. model. onthemixed-effects species) based with plantdiversity (0-9 plantsinthesame reproductive of vegetative or This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article J. vulgaris Final modelsequation illustrating structural the strength and direction ofthe Effect of the diversity of theneig diversity of ofthe Effect Effect of diversity of the neighbouring co oftheneighbouring ofdiversity Effect P plants.grey Dark greyand denote positive light arrows and negative significant plants. Means ± between-plot SE are shown. shown. are Means± SE plants. between-plot < 0.05. The strength of the direct path <0.05.Thestrength ofthedirect plants.Means± SEar between-plot bundance associated tothefocal associated bundance and its significance isdenotedas*** and itssignificance es show non-significant effects at effects es shownon-significant -1 dw) in the leaves of the vegetative and reproductive and ofthevegetative dw)intheleaves of the neighbouring community, focal plant community, focal of theneighbouring ecies) based on the mixed-effects model. model. onthemixed-effects based ecies) hbouring community on the aboveground plant community plant ontheaboveground hbouring of the vegetative and reproductive focal and reproductive ofthevegetative ifoline-type (Er), senecionine-type (Sn), and senecionine-type (Er), ifoline-type proportional to standardized path coefficients pathcoefficients tostandardized proportional s corresponds to the path coefficient. tothepathcoefficient. s corresponds plot. Lines indicate a significant relationship Lines asignificant plot. indicate mmunity on the total PA concentration and totalPAconcentration mmunity onthe e shown. Lines indicate a significant asignificant indicate Lines e shown. Lines relationship asignificant indicate
vegetative and reproductive and reproductive vegetative P > 0.05 thatwere >0.05 P < 0.001; ** < J. P < J. Fig. 1. Fig. 1.
This article is protected by copyright. All rights reserved. copyright. This articleis protectedby
Accepted Article Community diversity characteristics Community Community Community Community height cover characteristics concentration concentration J. vulgaris J. vulgaris JV shoot biomass JV PA JV N JV
Generalist Generalist herbivore Arthropod community Arthropod on Carnivorous arthropods J. vulgaris Specialist herbivore
Fig. 2. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article No. of carnivorous arthropods No. of generalist herbviores No. of specialized herbviores No. of arthropods per plant per plant per plant per plant 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0246810 Vegetative Vegetative Plant diversity J. vulgaris J. 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.5 1.0 1.5 2.0 10 15 20 25 30 10 15 20 25 30 0 5 0 5 0246810 Reproductive Plant diversity Plant
J. vulgaris
Fig. 3. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article C:N ratio Nitrogen (%) Root biomass (g) Shoot biomass (g) 10 15 20 25 30 35 0 1 2 3 4 0 1 2 3 4 0 1 2 3 0 5 0246810 Vegetative Plant diversity J. vulgaris 10 15 20 25 30 10 15 20 25 30 35 10 0 1 2 3 4 5 0 5 0 5 0 2 4 6 8 0246810 Reproductive Plant diversity J. vulgaris
Fig. 4. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby AcceptedSp-type PAs concentration Sn-type PAs concentration Er-type PAs concentrationArticleJb-type PAs concentration Total PA concentration 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.3 0.6 0.9 1.2 1.5 1.8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 6 0 2 4 6 8 0246810 Vegetative Plant diversity J. vulgaris J. 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.3 0.6 0.9 1.2 1.5 1.8 4 6 8 0 2 0 1 2 3 4 5 6 0246810 Reproductive Reproductive Plant diversity J. vulgaris J.
Fig. 5. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Community Community Accepted Community Article diversity diversity
0.39** 0.37** 0.30* characteristics characteristics 0.39** Community Community Community Community Community Community Community Community Community Community height height cover cover 0.27* -0.54*** -0.50*** -0.33** -0.17* 0.31* Vegetative 0.38*** 0.68*** characteristics characteristics Reproductive concentration concentration concentration concentration J. vulgaris J. vulgaris vulgaris J. JV shoot JV JV shoot JV biomass biomass JV PA JV PA JV N JV N J. vulgaris 0.45*** 0.41** 0.49*** 0.22* J. vulgaris J.
0.26* 0.71*** Generalist Generalist Generalist Generalist herbivore herbivore 0.44** Arthropod community Arthropod Arthropod community Arthropod -0.40*** on on 0.28* Carnivorous Carnivorous arthropods arthropods J. vulgaris J. vulgaris -0.35* Specialist Specialist herbivore herbivore 0.67***