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E ec s o soil abio ic ac o s and plan chemical de ences on seed p eda ion on sea
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ennel (C i hmum ma i imum)
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Running i le: Soil and seed p eda ion
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Xoaquín Mo ei a1*, Ignacio M. Pé ez-Ramos2, Luis Ma ías3, Ma a F ancisco1, Albe o
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Ga cía-González3, Raquel Ma ins-Nogue ol3, Ca la Vázquez-González1, Luis Abdala-
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Robe s4, and Jesús Camb ollé3
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1Misión Biológica de Galicia (MBG-CSIC), Apdo. 28, 36080 Pon e ed a, Galicia,
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Spain
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2Ins i u o de Recu sos Na u ales y Ag obiología de Se illa (IRNAS-CSIC), P.O. Box
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1052, 41080 Se illa, Andalucía, Spain
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3Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de
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Se illa, P o eso Ga cía González s/n, E-41012, Se illa, Spain
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4Depa men o T opical Ecology, Au onomous Uni e si y o Yuca an, Apa ado Pos al
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4-116, I zimna. 97000. Me ida, Yuca an, Mexico
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*Co esponding au ho :
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Email: [email p o ec ed]
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Phone Numbe : +34 986854800 Fax Numbe : + 34 986841362
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Pos p in o : Plan and Soil (2021) h ps://doi.o g/10.1007/s11104-021-04994-x
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ABSTRACT
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Aims: Soil abio ic ac o s commonly in luence plan de ensi e ai s by shaping he
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cos s o de ence p oduc ion and hese bo om-up e ec s on plan s can in u n a ec
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insec he bi o y. Howe e , ew s udies ha e disen angled di ec and indi ec e ec s o
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soil abio ic ac o s on plan de ences and insec he bi o y.
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Me hods: To add ess his gap, we es ed he in luence o soil abio ic ac o s on seed
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p eda ion ia changes in plan de ences o sea ennel (C i hmum ma i imum), a
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common coas al plan in sou he n Spain whose seeds a e consumed by specialis
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ca e pilla s (Ae hes species). To his end, we es ima ed seed p eda ion on plan s ac oss
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se e al sea ennel popula ions, as well as measu ed di e en ypes o pu a i e
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chemical seed de ences (phenolics, e penes) and soil abio ic ac o s (mac o- and
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mic o-elemen s, physiochemical a iables).
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Resul s: We ound a posi i e associa ion be ween seed chemical de ences ( e penes
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such as α- hujene, α-pinene, β-pinene, β-my cene, α- e pinene, y- e pinene, and
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hymol me hyle he ) and seed p eda ion. In addi ion, a ew mac o- and mic o-
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elemen s such as Ca, S and S nega i ely co ela ed wi h seed de ences ( e penes);
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o he mac o- and mic o-elemen s o physiochemical a iables had no de ec able
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associa ion wi h de ences. Despi e obse ed e ec s o soil abio ic ac o s on de ences
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and o he la e on seed p eda ion, he e was no de ec able indi ec e ec o soil
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abio ic ac o s on seed a ack.
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Conclusions: Ou indings sugges ha a ia ion in a ew key soil mac o- and mic o-
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elemen s in hese coas al en i onmen s can exe an impo an in luence on seed
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chemical de ences in sea ennel, wi h po en ial consequences o in e ac ions be ween
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sea ennel and seed p eda o s.
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Keywo ds: Ae hes species, coas al en i onmen , he bi o y, phenolics, sea ennel, soil
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mac o- and mic o-elemen s, e penes
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INTRODUCTION
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Insec he bi o y is an ancien and widesp ead in e ac ion (Labandei a 2007). O e
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e olu iona y ime, a s iking di e si y o phy ophagous insec s has a isen and hese all
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in o di e en eeding guilds (e.g. lea chewe s and mine s, cell-con en eede s,
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pie cing-sucking he bi o es, oo eede s, gall-make s, and seed p eda o s; Ma quis
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1992), which ha e a ying nega i e consequences on plan g ow h and ep oduc ion
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(Ma on 1998). Amongs hese g oups, p e-dispe sal seed p eda o s ha e pa icula ly
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nega i e consequences o plan ep oduc i e ou pu and popula ion dynamics (Kolb e
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al. 2007), and many o hese he bi o es exhibi high die a y specializa ion (Abdala-
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Robe s and Mooney 2013; Chen and Moles 2018; G ipenbe g e al. 2019).
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Acco dingly, p e-dispe sal seed p eda o s ha e li e cycles ha a e highly synch onized
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wi h plan ep oduc i e phenology (Diniz and Mo ais 2002; No o ny and Basse 2015)
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and o en exhibi co-e olu iona y a ms aces wi h hei hos plan s (Be enbaum and
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Zange l 1998; Thompson 2005).
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Resea ch on plan -he bi o e in e ac ions has his o ically ocused on plan
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de ences and he mechanisms by which he po ency and di e si y o de ensi e ai s has
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e ol ed as a means o end o he bi o es o educe hei consump ion (Simms and F i z
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1990; Ag awal 2007; Fu uyma and Ag awal 2009). In u n, s udies ha e also shown ha
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quan i a i e and quali a i e a ia ion in plan physical and chemical de ences play an
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impo an ole in de e mining he isk and in ensi y o he bi o y (Feeny 1976; Ma quis
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1992), as well as in shaping insec he bi o e communi ies (Richa ds e al. 2015; Salaza
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e al. 2016). Unde s anding hese bo om-up e ec s o plan de ences on phy ophagous
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insec s hus ep esen s a undamen al challenge o explaining in a- and in e -speci ic
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a iabili y in he bi o y, one ha also equi es emb acing he complex na u e o plan
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de ensi e pheno ypes by simul aneously s udying mul iple, o en co ela ed, ai s
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(Mo ei a e al. 2020a; Quijano-Medina e al. 2021).
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O he well-known sou ces o bo om-up con ol in he bi o y a e abio ic ac o s,
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including soil mac o- and mic o-elemen s (Coley e al. 1985; Fine e al. 2004), and
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wa e o ligh a ailabili y (Gu b od e al. 2011; Abdala-Robe s e al. 2014; Balla é
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2014). Plan de ence heo y pos ula es ha , since he cos o eplacemen o damaged
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issues by he bi o es is highe in esou ce-limi ed en i onmen s (e.g. low a ailabili y o
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soil mac o- and mic o-elemen s, high soil salini y), plan s should exhibi highe le els
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o de ences when esou ces a e sca ce ( e iewed by S amp 2003). By inc easing plan
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de ences, soil abio ic ac o s can in u n nega i ely a ec he bi o e hos plan choice o
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eeding beha iou (Abdala-Robe s e al. 2016a; Mo ei a e al. 2018), leading o
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dec eased he bi o e damage. To da e, howe e , s udies disen angling di ec and indi ec
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e ec s o soil abio ic ac o s on plan de ences and he bi o y a e sca ce (bu see Dalling
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e al. 2011; Mo ei a e al. 2018).
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Sea ennel (C i hmum ma i imum L.) is a common halophy e he b in coas al
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habi a s h oughou Wes e n Eu ope, and is a acked by seed-ea ing specialis
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ca e pilla s o he genus Ae hes spp. These insec s a e highly common on sea ennel
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plan s, esul ing in up o 80% o seeds a acked a some si es. In addi ion, he coas al
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si es whe e sea ennel g ows exhibi a iable bu gene ally low a ailabili y o mac o-
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and mic o-elemen s, as well as high soil salini y, alkalini y, and physical s ess (e.g.
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wind exposu e). In he p esen s udy, we sampled sea ennel plan s om se en
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popula ions loca ed in he sou h o Ibe ian Peninsula, spanning a wide ange o soil
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condi ions ( om sandy beaches o s eep cli s). A each popula ion, we es ima ed seed
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p eda ion, and measu ed se e al ypes o seed chemical de ences (phenolics, e penes)
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as well as associa ed soil abio ic ac o s (mac o- and mic o-elemen s, physiochemical
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a iables). Phenolic compounds and e penes a e pu a i e de ensi e compounds ha a e
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oxic and de e en o a b oad ange o phy ophagous insec s, and a e also associa ed
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wi h in e ac ions in ol ing specialis he bi o es (Mi hö e and Boland 2012). We es ed
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whe he chemical de ences co ela e wi h seed p eda ion, i soil abio ic ac o s co ela e
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wi h seed de ensi e ai s, and whe he any such associa ions be ween soil abio ic
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ac o s and seed de ences indi ec ly a ec seed p eda ion. We expec ed ha de ence
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le els would nega i ely co ela e wi h he bi o y (signalling esis ance agains seed
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p eda ion ac oss popula ions), and ha plan s om si es wi h lowe esou ce a ailabili y
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and highe soil- ela ed abio ic s ess would be mo e chemically de ended, and in u n
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su e lowe seed p eda ion. By add essing mul iple plan de ences simul aneously,
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p o iding a comp ehensi e assessmen o soil abio ic ac o s, and es ing o bo h di ec
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and indi ec soil e ec s on seed p eda ion, his s udy con ibu es o a mo e nuanced
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unde s anding o bo om-up e ec s o soil abio ic ac o s and plan de ences on p e-
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dispe sal seed p eda ion.
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MATERIAL AND METHODS
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Na u al his o y
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Sea ennel (C i hmum ma i imum L.) is a pe ennial halophy e he b dis ibu ed
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h oughou he wes e n coas s o Eu ope, No h A ica and he Black Sea. I lowe s
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om June o Sep embe , and ui s ma u e om Sep embe o Decembe . Plan s
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ypically bea 100-200 in lo escences a he peak o he lowe ing season. Each ui
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con ains a single d y seed om 4–10 mm long. Along i s dis ibu ion, sea ennel is
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ound in bo h sandy and ocky beaches, as well as cli s, and g ows equen ly in sandy
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soils, wi h low a ailabili y o mac o- and mic o-elemen s and high alkalini y and
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salini y (Meo -Du os and Magné 2009).
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Some o he mos impo an insec he bi o es on his plan a e p e-dispe sal seed
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p eda o s o he genus Ae hes species (Lepidop e a: To icidae) (e.g. A. bilbaensis, A.
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ancillana, A. eichle i), which specialize on se e al species o Apiaceae (mainly C.
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ma i imum and Ca um e icilla um). The adul emale mo h bo es a small hole h ough
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he coa o imma u e ui s o lay eggs in June-July. Mo h la ae hen g ow inside ui s
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eeding on he seeds and lea e he ma u e ui by d illing an exi hole in Sep embe -
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Oc obe . Then, la ae bo e in o he s ems and hibe na es un il he ollowing g owing
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season.
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Field sampling
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In mid-Sep embe , when plan s bea bo h imma u e and ma u e ui s, we su eyed
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se en popula ions o sea ennel in sou he n Po ugal and sou he n Spain (Fig. 1). The
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si es sampled had di e en soil cha ac e is ics and habi a opog aphies, including sandy
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o ocky beaches, and cli s. Popula ions we e sepa a ed by 13 o 785 km, and a y wo-
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old in annual p ecipi a ion and 1.2 ºC in annual mean empe a u e. Likewise, soil
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abio ic ac o s, including mac o- and mic o-elemen s, also a y conside ably ac oss
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hese si es (Table S1 in he Supplemen a y Ma e ial). Each popula ion was comp ised o
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a leas 30 adul sea ennel plan s.
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A each si e, we selec ed 11-12 adul ( ep oduc i e) plan s (n = 83) o simila
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heigh ( ange: 10-56 cm; 44.59 ± 1.62 cm [mean ± SE]). Plan s we e sepa a ed by a
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leas 2 m, and displayed, on a e age, 186.3 ± 15.1 (SE) umbels and 351.6 ± 20.6 seeds
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pe umbel. Fo each plan , we collec ed se en umbels wi h imma u e ui s, placed hem
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in ice a -10°C, and anspo ed hem o he labo a o y. Then, we opened he ui s o
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ob ain he seed, and g ound he seeds wi h liquid ni ogen and s o ed hem a -80ºC o
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chemical analyses. Du ing sampling, we ook special ca e o only collec undamaged
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imma u e ui s such ha chemical de ence measu emen s ep esen ed a ough p oxy o
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cons i u i e seed de ences ( he in luence o sys emic induc ion i o he umbels in he
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same plan we e a acked canno be disca ded). In addi ion, o assess ui p eda ion, we
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collec ed om he same plan s se en mo e umbels in which all o mos ui s we e
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ma u e and we e placed in pape en elopes and anspo ed o he labo a o y whe e
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la ae con inued eeding and comple ed hei de elopmen . These samples we e used o
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es ima e seed p eda ion (see nex ).
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Es ima ion o seed p eda ion
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Fo each plan , we coun ed he o al numbe o ma u e ui s (i.e. seeds) in each umbel
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and calcula ed he p opo ion o seeds a acked (numbe o a acked seeds / o al numbe
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o ma u e seeds collec ed pe plan ), i.e. “seed p eda ion” he ea e . Seed p eda ion was
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o e whelmingly caused by la ae o Ae hes species (> 95% o cases; J. Camb ollé, da a
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om his s udy).
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Quan i ica ion o seed chemical de ences
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Phenolic compounds a e eeding de e en s agains insec seed p eda o s ound in many
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plan axa (Dalling e al. 2020), including Apiaceae species (Be enbaum 2001). B ie ly,
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we ex ac ed phenolic compounds om imma u e seeds using 20 mg o d y ma e ial
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(o en-d ied o 48 h a 40°C) wi h 0.25 mL o 70% me hanol in an ul asonic ba h o
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15 min, ollowed by cen i uga ion (Mo ei a e al. 2014). We hen ans e ed he
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ex ac s o ch oma og aphic ials o pe o m phenolic p o iling. Fo phenolic compound
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iden i ica ion, we used an ul a-pe o mance liquid ch oma og aphy coupled wi h
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elec osp ay ioniza ion quad upole (The mo Dionex Ul ima e 3000 LC) ime-o - ligh
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mass spec ome y (UPLC-Q-TOF-MS/MS) (B uke Compac ™) (Mo ei a e al.
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2020b). We pe o med ch oma og aphic sepa a ion in a B uke UHPLC In ensi y Solo 2
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C18 2.1 × 100 mm 1.7 μm po e size column using a bina y g adien sol en mode
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consis ing o 0.1% o mic acid in wa e (sol en A) and ace oni ile (sol en B). We
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used he ollowing g adien : 3% B (0–3 min), om 3% o 25% B (3–10 min), om 25% o
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80% B (10–18 min), om 80% o 100% B (18–22 min), and held a 100% B un il 24 min.
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The injec ion olume was 3 µL, he low a e was es ablished a 0.3 mL min-1 and
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column empe a u e was con olled a 35°C. We ope a ed MS analysis in a spec a
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acquisi ion ange om 50 o 1200 m/z. We used nega i e (-) ESI modes unde he
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ollowing speci ic condi ions: gas low 8 l/min, nebulize p essu e 38 psi, d y gas 7 L
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min-1, and d y empe a u e 220 °C. We se capilla y and end pla e o se o 4500 and
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500 V, espec i ely. We pe o med Tandem Mass Spec ome y (MS/MS) analysis
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based on he p e iously de e mined accu a e mass and e en ion imes and agmen ed
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by using di e en collision ene gy amps o co e a ange om 15 o 50 eV. We
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eco ded ch oma og ams a 330 nm. We iden i ied indi idual compounds based on he
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da a ob ained om he s anda d subs ances o published li e a u e including e en ion
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imes, λmax, ([M–H]−), and majo agmen ions. We only iden i ied phenolic
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compounds om wo g oups: la onoids (N = 7) and hyd oxycinnamic acids (N = 3).
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Fo phenolic compound quan i ica ion, we injec ed 10 µL o each sample (using he
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same column and condi ions men ioned abo e) in an UHPLC (Nexe a LC-30AD;
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Shimadzu) equipped wi h a Nexe a SIL-30AC injec o and one SPD-M20A UV/VIS
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pho odiode a ay de ec o (Mo ei a e al. 2018). We quan i ied la onoids as u in
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equi alen s and hyd oxycinnamic acids as e ulic acid equi alen s (Mo ei a e al. 2018).
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We achie ed he quan i ica ion o hese phenolic compounds by ex e nal calib a ion
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using calib a ion cu es a 0.25, 0.5, 1, 2 and 5 μg mL-1. We exp essed phenolic
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compound concen a ions in mg g-1 issue on a d y weigh basis and analysed plan -le el
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da a o o al concen a ion o phenolics and by ype o phenolic compounds (see
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s a is ical analyses ahead).
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We also quan i ied e penes, which a e simila ly conside ed pu a i e chemical
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de ences agains insec seed p eda o s in many plan axa (Dalling e al. 2020),
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including Apiaceae species (Be enbaum 2001). Fo his, we ex ac ed compounds om
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imma u e seeds using 300 mg o g ound esh ma e ial wi h 1 mL o 70% me hanol in
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an ul asonic ba h o 20 min and s o ed samples a 4°C o 24 h. We also added
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dodecane (Me ck, #1.09658.0005) as he in e nal s anda d solu ion (100 ppm o
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dodecane in n-hexane). We injec ed he samples (1 μL) on o a gas ch oma og aph (GC,
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The mo Finnegan T ace GC Ul a, Wal ham, MA, USA) wi h a mass spec ome e
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(MS) de ec o ha was i ed wi h a 30 m × 0.25 mm × 0.25 μ ilm hickness ZB-5MSi
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(Phenomenex, UK) in single ion moni o ing mode (SIM: m/z 68, 69, 77, 79, 92, 93, 94,
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105, 119, 121, 136, 148, 161, 175) used o make isible known e pene agmen s. The
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GC was ope a ed in spli mode (50 mL min-1) wi h helium as he ca ie gas ( low a e 1
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mL min-1). The GC o en empe a u e p og am was: 2 min hold a 60°C, 10°C min-1
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amp o 70°C, 15 min hold a 70°C, 5°C min-1 amp o 130°C, 30°C min-1 amp o
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250°C, and 1 min hold a 250°C. We iden i ied e penes compa ing hei Ko á s
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indices, calcula ed ela i e o he e en ion imes o a se ies o n-alkanes (C8-C20,
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Sigma-Ald ich, Me ck KGaA, Da ms ad , Ge many) analysed unde he same
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ch oma og aphic condi ions, wi h hose epo ed in he li e a u e (Tsouka ou e al. 2001;
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Nabe e al. 2017). We only iden i ied mono e penes. Fo each plan , we es ima ed he
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amoun o e penes by using no malized peak a eas pe d y weigh . The no malized
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peak a ea pe d y weigh o each compound was ob ained by di iding hei in eg a ed
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pa icula ly o specialis he bi o es (e.g. Cas illo e al. 2014; Abdala-Robe s e al.
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2016a; Ochoa-López e al. 2020). One possible explana ion is ha e penes (including
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ola ile compounds) se e as hos inding cues by Ae hes o iposi ing emales, as shown
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o o he seed-ea ing species o To icidae o which speci ic mono e penes ha e been
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shown o a ac o iposi ing emales (e.g. Wea ing and Hu chins 1973; Su he land e al.
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1977), and simila indings ha e been epo ed o o he g oups o specialis insec
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he bi o es such as Coleop e a (e.g. ba k bee les, E bilgin and Ra a 2000; Seybold e al.
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2006). In addi ion, hese ca e pilla s may ob ain physiological o immunological
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bene i s om eeding on hos plan oxins ( e iewed by E b and Robe 2016),
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including e penes (e.g. Bece a 1997) as well as o he ypes o compounds such
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u anocouma ins which a e also common in he case o Apiaceae (e.g. Ca oll e al.
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1997; Ca oll and Be enbaum 2006). These bene i s include boos ed immunological
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esponses agains pa hogens (e.g. Singe e al. 2014; Ba hel e al. 2016; Ga ey e al.
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2021) as well as de ence agains pa asi oids and p eda o s ia me aboli e seques a ion
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(Ka sanis e al. 2016; Kelly and Bowe s 2018; Ochoa-López e al. 2020). I should be
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no ed, howe e , ha al hough we used only undamaged seeds o chemical analyses,
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sys emic induc ion due o a ack on o he umbels o he same plan could ha e esul ed
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in highe chemical de ences in undamaged seeds (Abdala-Robe s e al. 2016b; Mo ei a
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e al. 2018), hus explaining a posi i e co ela ion be ween seed p eda ion and de ences
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( e penes). Pas s udies ha e ound e penes o be induced upon eeding by o he
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To icidae species (e.g. He n and Do n 2002; Giacomuzzi e al. 2016) and spa ial
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a ia ion in he bi o e p essu e could be an impo an d i e o popula ion a ia ion in
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sea ennel de ences as shown in o he plan axa in Apiaceae (Be enbaum and Zange l
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1998) o o he amilies (Züs e al. 2012; Abdala-Robe s e al. 2016b). Un o una ely,
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he me hodology used in he p esen s udy does no allow us o di e en ia e be ween
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hese wo explana ions o unambiguously assess bo om-up e ec s o plan de ences.
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Fu he wo k in ol ing sampling me hodologies in which he iming o seed sampling is
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conduc ed p io o he onse o seed p eda ion o gain insigh in o how p e-exis ing
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(cons i u i e) de ence le els a ec ( om he bo om-up) seed p eda ion. A he same
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ime, expe imen al manipula ion o he bi o y in si u o in g eenhouse condi ions,
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coupled wi h ca e pilla bioassays (including seques a ion o seconda y me aboli es)
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a e needed o unde s and he consequences o plan induced esponses o his
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in e ac ion.
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While phenolics and e penes a e commonly s udied as de ences in he con ex
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o plan -he bi o e in e ac ions, hey may also se e o o he unc ions, pa icula ly in
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he con ex o abio ic o physical s ess. Fo example, phenolics a e exp essed in highe
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le els unde condi ions o high ligh a ailabili y o p o ec lea es om damaging e ec s
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o excess ligh (Abdala-Robe s e al. 2014; e iewed by Balla é 2014). Likewise,
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e penes ha e also been shown o be in ol ed in plan p o ec ion agains ex eme abio ic
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condi ions such as d ough o high empe a u es (e.g. Llusià and Peñuelas 1998),
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indica ing also a ole in abio ic ole ance and s ess esponses. In addi ion, ecen wo k
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shows ha sal -s essed plan s can p ime physiological esponses o be e cope wi h
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salini y s ess in neighbou ing plan s ia ai bo ne e penes (Capa o a e al. 2018).
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Based on da a cu en ly a hand in ou s udy sys em, i is unclea whe he hey a e
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known o unc ion as de ences in his sys em and especially agains he s udied seed
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p eda o . In his sense, manipula ions o abio ic ac o s such as salini y, empe a u e, o
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soil humidi y could be coupled wi h manipula ions o ocal soil nu ien s o unde s and
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he join in luences and in e ac ions be ween soil esou ces and o he abio ic ac o s
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po en ially in luencing sea ennel chemical de ences.
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Despi e soil mac o- and mic o-elemen s nega i ely co ela ed wi h seed de ences
425
( e penes) and hese me aboli es posi i ely co ela ed wi h seed p eda ion, he e was no
426
de ec able indi ec associa ion be ween soil abio ic ac o s and seed p eda ion. Recen
427
wo k o ou in es iga ing indi ec e ec s o soil and clima ic ac o s on he bi o y ia
428
plan de ences in o he plan axa has shown mixed e idence. Fo example, in
429
ag eemen wi h p esen indings, p ecipi a ion nega i ely a ec ed lea pubescence and
430
posi i ely a ec ed lea -chewe he bi o y in wild co on, bu he e was no indi ec e ec
431
o p ecipi a ion on he bi o y (Abdala-Robe s e al. 2019). Howe e , o English oak
432
(Que cus obu ) we ound ha empe a u e and p ecipi a ion we e nega i ely associa ed
433
wi h lea and seed de ences ( espec i ely) and his led o posi i e indi ec e ec on seed
434
p eda ion (Mo ei a e al. 2020b); likewise soil physical p ope ies (e.g. po osi y)
435
posi i ely co ela ed wi h oak chemical de ences and his led o an indi ec nega i e
436
e ec on lea he bi o y (Mo ei a e al. 2018). Resul s hus a appea o be sys em-
437
speci ic depending on he plan axa s udied o ype o de ences o abio ic ac o looked
438
a , hus wa an ing mo e s udies be o e gene al ules o he s eng h o indi ec abio ic
439
e ec s on he bi o y can be in e ed. Iden i ying he mos impo an abio ic ac o s
440
a ec ing plan de ences in a gi en sys em (e.g. limi ing mac o- and mic o-elemen s o
441
soil physical p ope ies), as well as key physical o chemical de ences wi h known
442
impac s on associa ed phy ophagous insec s (e.g. specialized chemical de ences agains
443
ocal insec he bi o es o b oad-spec um me aboli es agains di e se gene alis insec s)
444
a e needed in o de o achie e mo e ocalized and obus es s o abio ic o cing indi ec
445
e ec s on he bi o y.
446
We en ision a couple o a enues o u u e esea ch on abio ic bo om-up e ec s
447
on he bi o y in sea ennel as well as o he plan axa. Fi s , expanding esea ch o
448
include a g ea e numbe o popula ions eplica ed ac oss di e en coas al habi a s in
449
19
o de o assess spa ially a ying abio ic ac o s ha cause a ia ion in he bi o y.
450
Accoun ing o o he abio ic ac o s such as clima ic o mechanical damage due o wind
451
is also wa an ed o achie e a mo e comp ehensi e e alua ion o abio ic sou ces o
452
spa ial a ia ion in he bi o y. Second, conduc ing wi hin- and among-popula ion
453
manipula i e assessmen s o mac o- and mic o-elemen e ec s (mimicking obse ed
454
a ia ion in si u) on chemical de ences, bo h cons i u i e and induced, unde con olled
455
and/o in si u condi ions. These es s would also in ol e measu ing e ec s on
456
he bi o y, namely iden i ying key me aboli es ( ola ile o non- ola ile) in luencing
457
seed p eda o hos plan p e e ence and seed consump ion, as well as es ing o indi ec
458
e ec s o abio ic manipula ions on hese he bi o e esponses.
459
460
ACKNOWLEDGEMENTS
461
Commen s by Ma in Agui ebengoa and wo anonymous e iewe s helped o imp o e
462
he manusc ip . We a e g a e ul o Bea iz Lago-Núñez and Edua do Gu ié ez-
463
González o hei help wi h analyses o soil a iables and plan de ences.
464
465
FUNDING
466
This esea ch was inancially suppo ed by wo g an s om he Spanish Minis y o
467
Science, Inno a ion and Uni e si ies (RTI2018-099260-A-I00 o JC and RTI2018-
468
099322-B-I00 o XM). XM and IMPR we e inancially suppo ed by he Ramón y Cajal
469
Resea ch P og amme (RYC-2013-13230 and RYC-2013-13937, espec i ely).
470
471
AUTHOR CONTRIBUTIONS
472
Fo mula ed he idea o he manusc ip : XM, JC, LAR. Designed he expe imen : JC,
473
IMPR, LM. Pe o med he ield sampling: JC, IMPR, LM. Pe o med plan and soil
474
20
measu emen s: XM, JC, IMPR, LM, RMN, AGG, MF. Con ibu ed eagen s/ ools: XM,
475
JC. Analysed he da a: XM, CVG. W o e he i s d a o he manusc ip : XM.
476
Con ibu ed c i ically o he w i ing: LAR, JC, IMPR, LM.
477
478
ETHICS DECLARATION
479
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal
480
ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .
481
482
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483
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618
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32
FIGURE LEGENDS
732
733
Figu e 1. Map showing he loca ion o he se en popula ions o C i hmum ma i imum
734
sampled along he coas s o sou he n Po ugal and sou he n Spain.
735
736
Figu e 2. Diag am showing esul s om a piece-wise s uc u al equa ion model es ing
737
o di ec associa ions among soil abio ic ac o s and seed de ences, and among seed
738
de ences and seed p eda ion on C i hmum ma i imum indi iduals sampled om se en
739
popula ions. Soil abio ic ac o s ep esen z-sco e alues om a p incipal componen
740
analysis summa izing a sui e o a iables associa ed o soil mac o- and mic o-elemen s
741
and soil physico-chemical p ope ies (PC1 soil, PC2 soil and PC3 soil, see s a is ical
742
analyses). Seed de ences ep esen z-sco e alues om a p incipal componen analysis
743
summa izing a sui e o compounds associa ed o seed e penes and phenolics (PC1
744
de ences and PC2 de ences, see s a is ical analyses). Values nex o each a ow a e pa h
745
coe icien s (i.e. s anda dized pa ial eg ession coe icien s). Black a ows indica e
746
signi ican associa ions whe eas g ey a ows indica e non-signi ican associa ions.
747
Explained a iance: PC1 de ences (ma ginal = 0.15, condi ional = 0.24); PC2 de ences
748
= (ma ginal = 0.03, condi ional = 0.28); seed p eda ion = (ma ginal = 0.18, condi ional
749
= 0.59). Fishe 's C = 0.074, P = 0.964, AICc = 40.07.
750
751
752
753
754
755
756
