Performance of endophyte infected tall fescue in Europe and North America

RESEARCH ARTICLE Pe o mance o Endophy e In ec ed Tall Fescue in Eu ope and No h Ame ica Ka i Saikkonen 1 *, Timo hy D. Phillips 2 , S anley H. Fae h 3 , Rebecca L. McCulley 2 , I ma Saloniemi 4 , Ma jo Helande 1,4 1Na u al Resou ces Ins i u e Finland (Luke), Tu ku, Finland, 2Depa men o Plan and Soil Sciences, Uni e si y o Ken ucky, Lexing on, Ken ucky, Uni ed S a es o Ame ica, 3Depa men o Biology, Uni e si y o No h Ca olina, G eensbo o, No h Ca olina, Uni ed S a es o Ame ica, 4Depa men o Biology, Uni e si y o Tu ku, Tu ku, Finland *[email p o ec ed] Abs ac Human assis ed plan in asions om Eu ope o No h Ame ica ha e been mo e common han he e e se. We es ed endophy e-media ed pe o mance o all escue in pa allel h ee yea expe imen s in Eu ope and he USA using endophy e in ec ed and unin ec ed wild and cul i a ed plan s. Expe imen al plan s we e subjec ed o nu ien and wa e ea men s. Whe eas endophy e in ec ion inc eased all escue pe o mance in gene al, he e ec s o endophy es on plan g ow h and ep oduc ion a ied among plan o igins unde di e en en i- onmen al condi ions. Na u ally endophy e- ee Finnish cul i a ‘Re u’pe o med equally well as ‘Ken ucky-31’in bo h geog aphic loca ions. All Eu asian o igin plan s pe o med well in he US. In Finland, plan s es ablished well and bo h cul i a s su i ed o e he i s win e . How- e e , win e mo ali y o ‘Ken ucky-31’plan s was highe , pa icula ly in e ilized soils in he subsequen win e s. Ou esul s sugges ha all escue eco ype ‘Ken ucky-31’ ha lou ishes in No h Ame ica is poo ly adap ed o No he n Eu opean condi ions. In oduc ion Success ul plan in asions ha e been la gely unidi ec ional om Eu ope o No h Ame ica since he las Ice Age [1–4]. Since Eu opean colonis s disco e ed he Ame icas, human mig a- ion and inc eased global ade ha e acili a ed species in e change be ween he Old Wo ld and he Wes e n Hemisphe e, ia delibe a e in oduc ion o c op and o namen al plan s and unin- en ional in oduc ions, such as s owaways. Human-assis ed species in oduc ions ail, how- e e , o explain success ul na u aliza ion o Eu asian plan species in o Ame ica in gene al. The success ul in asion and subsequen na u aliza ion o alien species is o en linked o he cha ac- e is ics o he in ading species such as sel - e ili y, ep oduc i e s a egies, igo , pheno ypic plas ici y, and gene ic a ia ion ha allows passage h ough abio ic and bio ic en i onmen al il e s and p o ides highe po en ial o apid adap i e e olu ion o new condi ions. In con as , he lowe p obabili y o coloniza ion o No h Ame ican plan s o Eu ope may be a leas pa - ially explained by maladap a ion o No h Ame ican plan s o compa able clima e zones in Wes e n Eu ope ha ha e di e en pho ope iods. In gene al, clima es in Wes e n Eu ope ha PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 1/18 a11111 OPEN ACCESS Ci a ion: Saikkonen K, Phillips TD, Fae h SH, McCulley RL, Saloniemi I, Helande M (2016) Pe o mance o Endophy e In ec ed Tall Fescue in Eu ope and No h Ame ica. PLoS ONE 11(6): e0157382. doi:10.1371/jou nal.pone.0157382 Edi o : Leona d Simon an O e beek, Wageningen Uni e si y and Resea ch Cen e, NETHERLANDS Recei ed: Janua y 21, 2016 Accep ed: May 27, 2016 Published: June 10, 2016 Copy igh : © 2016 Saikkonen e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : The da a can be ound as supplemen a y ma e ial (S1 Da a.xls). Funding: The au ho s ha e no suppo o unding o epo . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . a e simila o No h Ame ican clima es a e a highe la i udes and hus ha e g ea e seasonal changes in pho ope iod [5–8,4]. The in asi eness o plan species may also be igh ly linked wi h p esence o mu ualis ic symbion s and o he se ice p o ide s such as pollina o s, o emp y niche and/o enemy escape in he ecipien en i onmen [9–15]. Fo example, ec omyco hizal ungi a e i ally impo an o he dis ibu ion o app oxima ely 6000 ee species including many dominan and econom- ically impo an species nea suba c ic ee-lines [16–18]. The impo ance o mic obes o plan communi y dynamics is, howe e , con ex dependen , inco po a ing mul ispecies in e ac ions and communi y eedbacks, modi ying niches o species compe ing and/o sha ing common esou ces [19]. Tempe a e g asslands a e pa icula ly ulne able o plan in asions because hey ha e a long his o y o being d i en by human ac i i ies such as ag icul u e [20,21]. In No h Ame ica, Eu opean se le s opened windows o plan in asions by apidly con e ing na i e g asslands and o es s in o a able lands and pas u es. The concomi an species in oduc ions we e la gely unidi ec ional om Eu ope o No h Ame ica. The g ea success o many Eu opean species in No h Ame ica may ha e been acili a ed by plan species adap a ions o hea y g azing by ca - le due o hei long- e m coexis ence wi h humans in he Eu asian en i onmen om whence hey o igina ed [3,20]. Be o e human se le s, he Holocene No h Ame ica g asslands had p i- ma ily been s uc u ed and main ained by i es. In he in e moun ain wes and G ea Plains, g azing by bison also main ained g asslands. Howe e , la ge a eas o No h Ame ican g ass- lands in eas e n No h Ame ica e ol ed unde he lack o hea y gazing p essu e un il humans s a ed o egula e i es, ein oduced he ho se, and in oduced ca le in he 1600s [3]. Inc eas- ing ag icul u e in conjunc ion wi h hese o he an h opogenic changes in ype, equency and in ensi y o dis u bances g ea ly acili a ed he es ablishmen and na u aliza ion o nonna i e plan species in o g asslands and des uc ion o na i e g assland plan communi ies in No h Ame ica [20,22]. Many g ass species we e acciden ally o in en ionally in oduced in o N. Ame ica, he la e because o hei ag onomic alue and high ole ance o a wide ange o bio ic and abio ic condi- ions. Alien g asses ha e become enacious in ade s o Na i e Ame ican g assland communi- ies. Inc easing e idence sugges s ha he success o some o he alien cool season g asses may be due, a leas in pa , o hei asymp oma ic and sys emic endophy ic ungi [11]. Tall escue [Schedono us phoenix (Scop.) Holub. ex. Lolium a undinaceum, syn. Fes uca a undinacea] pu hese sys emic g ass-endophy e symbioses on he map in he 1970s when li es ock diso de s associa ed wi h he ecen ly comme cialized all escue cul i a ‘Ken ucky 31’(KY-31) we e a ibu ed o myco oxins p oduced by he symbio ic Epichloë endophy e [23–26]. Tall escue was o iginally in oduced om Eu ope in he la e 1800’s, p obably as a con aminan in hay o packing ma e ials, and now i is he mos impo an cool-season g ass in he Uni ed S a es [27,28]. The obliga ely ou b eeding allohexaploid cul i a KY-31, which is commonly in ec ed wi h Epichloë coenophiala (Mo gan-Jones & W. Gams) C.W. Bacon & Scha dl (ex. Neo ypho- dium coenophialum Glenn, Hanlin & Bacon) endophy e, is adap ed o a wide ange o soil ypes, e ili y and pH, and ole an o mode a e cold and hea s ess. I has been and is s ill widely used o animal eed, lawns and u , soil s abiliza ion and wildli e ood plo s in humid a eas o he US. In 1977, KY-31 all escue composed app oxima ely 97% o all all escue u in he Uni ed S a es [29]. Simila o o he endophy e-g ass symbio a, endophy e in ec ion e- quency in all escue a ies, bu egional and he ba ium s udies ha e epo ed ha on a e age 60–80% o indi iduals a e in ec ed [30,31]. Since hen, his highly igo ous cul i a has become a enacious in ade o managed and unmanaged g asslands h ea ening he pe sis ence o na i e plan species di e si y h oughou he much o he eas e n Uni ed S a es [11,27,28,30]. Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 2/18 The compe i i e supe io i y o all escue is p omo ed by E.coenophiala endophy e [11] pa - icula ly in high nu ien ag o-en i onmen s [32]. Be o e he Eu opean se le s, only pa o he eas e n Uni ed S a es was hea ily g azed by na i e e eb a e g aze s [3,20]. In con as , he p esen all escue g een bel is unde high g azing p essu e by ca le [27], and acco dingly he an i-he bi o e ad an age p o ided by he endophy e o he hos has been sugges ed o cause a apid inc ease in endophy e equency in hos popula ions [30]. Because hese g ass endo- phy es a e e ically ansmi ed om ma e nal plan o o sp ing ia seeds, he in asi e g ass hos is a pheno ypic combina ion o plan and mic obe wi h a long co-e olu iona y his o y. In his symbiosis, he hos plan o en ecei es inc eased g ow h and ep oduc ion, and p o ec ion om pa hogens and he bi o es p o ided by ungal p oduced alkaloids [33–39], while he plan se es as a shel e , nu ien p o ide and ansmission aid o he ungus. Thus, g ass endophy e symbiosis is o en conside ed o be mu ualis ic [11,25]. Howe e , he empi ical e idence suppo ing he pu po ed g ass-endophy e mu ualism has his o ically been domina ed by a ew ag onomic and non-na i e model sys ems [38,39], pa ic- ula ly he all escue cul i a KY-31 and i s endophy ic pa ne E.coenophiala in he US. In con as , al hough his g ass species is widely dis ibu ed in Eu ope, in ec ed g asses do no de e animal g azing no a e hey compe i i ely dominan in na i e o human-modi ied eco- sys ems [31,32]. Tall escue is desc ibed as a species complex consis ing o h ee majo (Con i- nen al, Medi e anean and hizoma ous) mo pho ypes [40,41]. The Con inen al mo pho ype is sp ead o e no he n Eu ope and was also he ge mplasm s ock o he cool-season cul i a s in he US, including KY-31. Today, all escue is an inc easingly impo an o age g ass also h oughou Eu ope, and many cul i a s a e commonly used in ag icul u e. In his s udy, we examined i he success o all escue in he US is speci ic o hos popula- ion-le el geno ypic cha ac e is ics, endophy e in ec ion, o high nu ien ag o-en i onmen s. Speci ically we s udied in pa allel expe imen s in Finland and in he US how endophy e in ec- ion, plan o igin and nu ien and wa e a ailabili y a ec he g ow h and ep oduc i e capac- i y o all escue. The expe imen in Finland was si ua ed in a bo eal clima e a he same la i ude wi h Fai banks, Alaska, whe eas he expe imen in No h Ame ica is si ua ed in he empe a e zone a he same la i ude wi h A hens, G eece in sou he nmos Eu ope. In addi ion o wild plan s collec ed om h ee geog aphic loca ions in he no he nmos dis ibu ion ange o he species in Eu ope, we used wo cul i a s (KY-31 and ‘Re u’ om US and Eu ope, espec- i ely) in hese expe imen s. Fi s , we p edic ed ha plan s would pe o m bes in he clima ic and la i udinal en i onmen o which hey we e adap ed. Second, we hypo hesized ha a ia- ion in pe o mance would be lowe in cul i a s compa ed o g asses om wild popula ions because o lowe gene ic a ia ion due o b eeding. Consequen ly, he adap i e capaci y and pe o mance o cul i a s ac oss a wide ange o en i omen s should be lowe as well. In addi- ion o plan o igin, we p edic ed ha plan pe o mance would be a ec ed by he symbiosis wi h Epichloë endophy es. Pas li e a u e sugges s ha he mu ualism be ween E.coenophiala and KY-31 is s onges in high nu ien ag o-en i onmen s and may be a ypical o o he endo- phy e- all escue in e ac ions [38,39,42]. Thus, we p edic ed ha compa ed o KY-31, he bene- i s om endophy e in ec ion would be lesse in wild g asses e ol ed unde low he bi o y p essu e bu ins ead may depend mo e hea ily on nu ien a ailabili y in soils. Ma e ials and Me hods Plan ma e ial In Augus 2005, we collec ed seeds om na u al all escue popula ions a h ee geog aphic loca ions in he Bal ic Sea ha we e isola ed by app ox. 500 km om each o he : he island o Åland (A) (8 popula ions), island o Go land (G) (9 popula ions) and wes coas o Sweden (S) Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 3/18 (6 popula ions). We collec ed 10 o 50 plan indi iduals om each popula ion. The ield s udies did no in ol e endange ed o p o ec ed species and public igh o access in No dic coun ies allowed us o collec he samples wi hou speci ic pe missions. The p esence/absence o sys- emic endophy e in ec ion was checked by mic oscopic examina ion o h ee seeds om each indi idual plan [43]. All he s udied all escue popula ions had seed bo ne Epichloë coeno- phiala in ec ions a ying in in ec ion equency om 85–100% [31]. We combined all na u ally E- (unin ec ed) and E+ (in ec ed) seeds sepa a ely om each o he h ee geog aphic o igins (Åland, Go land and Sweden). In addi ion o plan s om na u al all escue popula ions, bo h E+ and E- ‘Ken uky 31’cul i a (KY-31) seeds we e ob ained om he Uni e si y o Ken ucky, as we e E- Finnish all escue cul i a ‘Re u’(R) seeds om he Plan Inspec ion Cen e, Seed Tes ing Depa men , Loimaa, Finland (www.e i a. i). Iden ical se s o seeds we e used in pa al- lel ield expe imen s in Finland and USA. Endophy e emo al A subse o he na u ally endophy e in ec ed (E+) seeds we e hea - ea ed by soaking hem in wa m wa e (56–57°C) o 10–20 minu es o kill he ungus while he seed emained iable. Using hese manipula i ely endophy e- ee (ME-) plan s, we aimed o sepa a e he e ec s o endophy e om he pheno ypic esponses o he endophy e-g ass symbio um, and/o es ima e i he plan pa ne is adap ed o he symbiosis du ing a coe olu iona y ela ionship [39]. We assume ha losing he long- ime mu ualis ic ungal pa ne would lead o lowe pe o mance o ME- plan s han na u ally endophy e- ee (E-) plan s in he same en i onmen . Pa allel ield expe imen s in Finland and USA The wo pa allel common ga den expe imen s we e ca ied ou in he ields o Tu ku Bo anical Ga den, Uni e si y o Tu ku, Finland (60°2600@N, 22°10019@E) and a he Uni e si y o Ken- ucky expe imen al a m in Eden Shale, Ken ucky, USA (38°32’22”N, 84°44’24”W). The ield si e in Finland is a he edge o he no he n dis ibu ion ange o na u al all escue popula- ions, while he Ken ucky ield si e is si ua ed in he hea o he in ensi e all escue cul i a ion a ea in USA–also known as he escue bel (h p:// o ages.o egons a e.edu/ all escuemonog aph/) and no a om whe e he all escue ha was e en ually de eloped in o he cul i a KY-31 was o iginally in oduced in he 1880’s (nea F enchbu g, Ken ucky). Bo h expe imen al ield si es had been in cul i a ion in he pas and we e illed wi hou nu ien en ichmen in he summe 2004. Win e empe a u es we e lowe in Finland and p ecipi a ion highe in Ken ucky (S1 Fig). Al hough win e s in Finland a e cha ac e ized by mon hs-long os empe a u es, g ea e snow co e may p o ide be e p o ec ion o plan s in he win e compa ed o Ken ucky. Acco ding o he Finnish Me eo ological Ins i u e's measu emen s (Finnish Me eo ological Ins i u e h p://www. mi. i/en), in 2006 he he mal g owing season was sligh ly longe han usual, and empe a u es we e highe . The sum o e ec i e empe a u e ( he sum o he posi i e di e ences be ween diu nal mean empe a u es and 5°C) eached new eco ds, and consequen ly he summe 2006 was he d ies e e expe ienced in Sou he n Finland. The expe imen al se -up was a andomized block design consis ing o 10 blocks, each including 4 plo s. In each o hese 40 plo s we plan ed g asses om ollowing geog aphic o igin —endophy e combina ions: na u al popula ions [Åland (A), Go land (G) and coas al Sweden (S)], cul i a [‘Ken ucky 31’(KY-31)] and endophy e in ec ion s a uses (E+, E-, ME-), and endophy e- ee (E-) cul i a ‘Re u’(R). In e e y plo , we had one plan om each o igin and endophy e s a us. Because cul i a ‘Re u’was always endophy e ee (E-), and in he Ken ucky expe imen we had only E+ and E- plan s o KY-31-o igin, he e we e 13 and 12 plan s in each Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 4/18 plo in Finland and in Ken ucky, espec i ely. The 40 plo s consis ing o 13 o 12 plan s summed up o o al o 520 plan s in Finland and 480 plan s in Ken ucky. Because na u ally occu ing and ag onomic ields o escue a e ne e single plan geno ypes, we chose a popula- ion-le el app oach o his s udy and did no quan i y indi idual plan geno ype esponses o ou ea men s. The all escue seeds we e ge mina ed on mois issue pape in Pe i dishes in a g eenhouse and plan ed in o indi idual po s wi h sand and pea mix u e 7 days a e ge mina ion. Plan s we e g own in he g eenhouse un il hey had 3 ille s and we e hen plan ed o he espec i e ields abou 0.5 m apa om each o he and om he edge o he plo in Augus 2004. The ou plo s in each block we e andomly designa ed o one o he ou ea men s: con- ol (C), wa e (W), nu ien (N), and combined wa e and nu ien (WN). The con ol ea - men ecei ed only ambien ain all. In Finland, W ea men plo s ecei ed 3 L o wa e applied o each plan sepa a ely h ee imes a week om June o Augus , o N ea men plan s 50 g o g anula N-P-K- e ilize (Nu men Y2, Kemi a KnowHow, N-P-K/20-6-6) was applied wo imes du ing he g owing seasons, and WN ea men ecei ed bo h wa e and nu ien applica ions. In Ken ucky, W ea men plan s ecei ed 3.8 L o wa e wice a week om Ap il o Oc obe , and e iliza ion consis ed o 50 kg/ha o N pe applica ion as u ea. The amoun o wa e applied co esponds o 350 mm p ecipi a ion which doubled he amoun o wa e ecei ed by plan s du ing he annual ea men pe iod in Ken ucky in bo h yea s and in Fin- land in 2005. In Finland, he W ea men quad upled he amoun o wa e ecei ed in he excep ionally d y summe o 2006. We acknowledge ha nume ous uncon olled di e ences be ween he wo expe imen al si es may con ound he compa ison o in e p e a ions om he wo expe imen s. To ake in o accoun he di e ences in nu ien con en s in soils, we analysed soil samples om he un ea ed (con ol) expe imen al plo s. The soil pH was 6.7 and 5.9, o al ni ogen 0.15% and 0.14%, phospho us 7 and 14 mg/kg, po assium 132 and 81 mg/kg, calcium 1800 and 2080 mg/kg and magnesium 208 and 120 mg/kg o Finland and Ken ucky si es, espec i ely. The wa e and nu ien ea men s we e applied o he expe imen s in wo g ow- ing seasons (2005 and 2006). The expe imen al a eas we e enced o p e en la ge e eb a es (e.g., abbi s, dee ) om dis u bing he expe imen al plan s. Howe e , smalle e eb a es (e.g., oles) and in e eb a es we e able o eely access he a ea. The space be ween he expe imen al plan s was ei he hand weeded o sp ayed wi h he bicide (glyphosa e Roundup1Bio) wo imes du ing he g owing season o p e en in e speci ic compe i ion be ween weeds and he expe imen al plan s. Response a iables In 2005, all he expe imen al plan s we e double-checked o e i y hei endophy e s a us. Fi s , we sampled a pseudos em om each plan o immunoblo assay o de ec monoclonal an i- bodies speci ic o Epichloë (Phy osc een Immunoplo Ki #ENDO7973, Ag inos ics, Wa kins- ille, Geo gia, USA). In addi ion, a he end o he summe , h ee seeds om each plan we e s ained [43] and endophy e in ec ions we e checked by mic oscopic examina ion. The endo- phy e s a us o ou and 35 plan s ou o 520 and 480 plan s we e eassigned o a co ec endo- phy e s a us in he Finland and Ken ucky ga dens, espec i ely. The su i al o he expe imen al plan s was eco ded du ing he h ee s udy yea s (2005– 2007). The ege a i e g ow h and ep oduc i e alloca ion o he plan s was obse ed in 2005 and 2006 a bo h s udy si es a he end o g owing season: he numbe o lowe heads was coun ed on a pe plan basis and he abo e g ound biomass o indi idual plan s was ha es ed by cu ing he ille s a he heigh o 5 cm abo e he g ound using a ice cu ing sickle. The bio- mass was hen d ied and weighed. Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 5/18 S a is ical analyses All he s a is ical analyses we e pe o med in he R en i onmen (R Co e Team 2012). The mo ali y o plan s was analysed using gene al linea model glm in R [44] wi h binomial dis i- bu ion and logi -link unc ion. The s a is ical model o su i al included endophy e s a us, nu ien ea men s (N: N and WN combined) and con ol ea men s (C: C and W combined), and plan o igins (cul i a ‘Re u’as baseline, KY-31, A, G, S) and hei in e ac ions. Plan biomass and lowe head coun s we e analyzed using gene al linea mixed models p o- g am lme in R. The model included s udy yea (2005, 2006), coun y (Finland, Ken ucky), endophy e in ec ion s a us (E+, E-, ME-), g ass o igin (KY-31, Re u, A, G, S), ea men s (C, W, N, WN) as ixed ac o s and block as a andom ac o . No mali y o biomass and lowe - head coun s was gained a e squa e oo ans o ma ion (as sugges ed by he Box-Cox analysis, ℓ= ½). Resul s Su i al Plan su i al was highe in Ken ucky han in Finland. None o he plan s died in Ken ucky, whils in Finland he dea h a e g adually inc eased om one plan in he i s win e o 21 and 19 plan s in he ollowing wo win e s. In Finland su i al was lowes in KY-31 (83%), while he o he o igins anged om 88% (Re u) o 98% (A) (compa ison be ween o igins: n = 520, x 2 = 28.1, d = 4, p<0.0001; Fig 1). Mo ali y was highe in ni ogen e ilized plan s (12.7% s. 2.3%, n = 520, x 2 = 18.7, d = 1, p<0.0001) ega dless o he endophy e in ec ion s a us o he plan (su i als s endophy e: x 2 = 0.16, d = 2, p = 0.92). In he logi model, su i al o e il- ized KY-31 plan s in Finland (in e ac ion NKY-31: z = 2.05, P <0.04) was lowes , bu in o he o igins e iliza ion did no a ec su i al (Table 1). G ow h and ep oduc ion Plan g ow h and ep oduc ion di e ed be ween he wo geog aphic loca ions and s udy yea s (Figs 2and 3, Tables 2and 3,S2 Fig). Du ing he i s g owing season (2005), he plan s in Fin- land we e smalle and p oduced ewe lowe heads (biomass mean ± S.E.: 297 ± 6, n = 506; lowe heads mean ± S.E.: 20 ± 1, n = 518) compa ed o plan s in Ken ucky (biomass mean ± S. E.: 353 ± 7, n = 480; lowe heads mean ± S.E.: 33 ± 1, n = 480). The di e ence was e en mo e s iking in he ollowing yea (2006), when he plan s in Ken ucky g ew 60% la ge and p o- duced wice as many lowe heads compa ed o he plan s in Finland (USA: biomass mean ± S. E.: 496 ± 13, n = 477; lowe heads mean ± S.E.: 69± 2, n = 456; Finland: biomass mean ± S.E.: 201 ± 6, n = 486; lowe heads mean ± S.E.: 35 ± 2, n = 497). I is no ewo hy ha in Finland in he excep ionally d y summe o 2006 (S1 Fig; Finnish Me eo ological Ins i u e h p://www. mi. i/en), some o he KY-31 plan s had died a e ha sh win e condi ions, and some o he su i ing KY-31 plan s g ew poo ly. G ow h and ep oduc ion in Finland The plan s in Finland we e signi ican ly la ge in 2005 compa ed o 2006 (Figs 2and 3) p oba- bly because o he excep ionally d y wea he condi ions in 2006. The combined wa e and nu ien ea men (WN) a ec ed simila ly he size o he plan s om he h ee wild Eu opean popula ions (A, G and S) in bo h s udy yea s: he biomass o he wild plan s was always highes o WN ea ed plan s (Figs 2and 4,Table 2). Howe e , he bio- mass o he wo cul i a s (KY-31 and R) was no in luenced by he ea men s, especially in he second s udy yea (Fig 4). Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 6/18 Plan o igin did no a ec biomass o he plan s in he i s s udy yea (2005), bu in 2006, cul i a KY-31 was he smalles , and cul i a ‘Re u’ he second smalles compa ed o plan s om he Eu opean wild popula ions (Fig 4). To al lowe head p oduc ion was 43% highe in 2006 compa ed o yea 2005. While all wild o igins ep oduced be e in 2006, his di e enced could no be de ec ed in cul i a s. On he con a y, KY-31 cul i a p oduced he highes numbe s o lowe heads in he i s s udy yea (2005), bu he lowes numbe o lowe heads in he yea 2006 (Fig 5,Table 3). The combined nu ien and wa e ea men a ec ed lowe head p oduc ion in he i s s udy yea 2005, when WN ea ed plan s p oduced mo e lowe heads han he plan s in he o he ea men s. Howe e , in he second s udy yea (2006), C and W ea men plan s p o- duced wice as many lowe s compa ed o nu ien ea ed (N and WN) plan s (Figs 2and 5). Fig 1. Mo ali y o all escue plan s in Finland in he end o he expe imen . Plan o igins: K = ‘Ken ucky 31’cul i a , R = ‘Re u’cul i a , A = wild ype om Åland, G = wild ype om Go land, S = wild ype om cos al Sweden. T ea men s: C = con ol, W = wa e applica ion, N = nu ien applica ion, WN = wa e and nu ien applica ion. The o igin- ea men in e ac ion is indica ed by *(see Table 1). doi:10.1371/jou nal.pone.0157382.g001 Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 7/18 This migh be pa ly explained by excep ionally wa m and d y summe condi ions limi ing nu ien cycling o soils and dissolu ion o applied g anula om he op-soils. G ow h and ep oduc ion in Ken ucky In he Ken ucky expe imen , plan s had 25% highe biomass and p oduced wice as many low- e s in he second s udy yea compa ed o i s s udy yea (Figs 2,3and 5). In he i s s udy yea (2005), plan s g ew sligh ly, bu no s a is ically signi ican ly, la ge on WN ea men plo s compa ed o o he ea men s (Figs 2and 4), bu p oduced equal numbe s o lowe heads on all he ea men s (Figs 2and 5). In he second s udy yea (2006), biomass o con ol plan s (C) was lowes , whe eas biomass in he N and WN ea men was 30–50% highe compa ed o o he ea men s (Yea N in e ac ion in Table 2), and he lowe head p oduc ion in he WN ea men was highe han in he C and W ea men s (Yea T ea N -in e ac ion in Table 3,Fig 5). Biomass o he wo cul i a s (Re u and KY-31) was highe compa ed o wild o igin plan s (A, G and S) in he i s s udy yea (2005), bu his di e ence was no de ec ed in he second s udy yea (2006), when all he plan s we e abou he same size (Yea O ig -in e ac ions o wild o igins Table 2,Fig 4). This sugges s ha he cul i a s we e less plas ic; he biomass was 15–20% and 40–60% highe in 2006 compa ed o 2005 in cul i a s and wild o igin plan s, espec i ely. The wild Eu opean o igin plan s g ew be e unde ea men s W, N and WN. Flowe head p oduc ion o he wo cul i a s (KY-31 and Re u) was clea ly supe io com- pa ed o wild o igin plan s (Table 3). In he second yea , he cul i a s p oduced wice as many lowe heads compa ed o he wild o igin plan s (Fig 5,Table 3). Endophy es The o e all e ec o endophy e in ec ion on all escue g ow h and ep oduc ion was signi i- can : E+ g asses ended o be la ge and ha e highe numbe o lowe heads compa ed o E- and ME- g asses (Figs 2and 3, Tables 2and 3). Table 1. Mo ali y o all escue plan s in Finland. Es ima e z p In e cep 2,20 2.948 0.0032 ** O ig K 1,88 1.499 0.1338 O ig A 1,17 1.130 0.2586 O ig G 1,88 1.499 0.1338 O ig S 17,37 0.013 0.9900 T ea N -0,46 -0.475 0.6346 O ig K: T ea N -2,92 -2.046 0.0408 * O ig A: T ea N 16,66 0.012 0.9904 O ig G: T ea N -1,42 -0.967 0.3336 O ig S: T ea N -16,46 -0.012 0.9905 Logi model o mo ali y in Finland as a unc ion o o igin (O ig) ( h ee wild popula ions and wo cul i a s: A=Åland island, G = Go land island, S = coas al Sweden, K = cul i a ‘Ken ucky-31’, Re u = cul i a ‘Re u’) and ea men s (T ea ) (Nu ien ea men s N = N and WN combined, con ol ea men s C = C and W combined) and hei in e ac ion. The Finnish cul i a ‘Re u’wi hou e iliza ion was used as he baseline o compa isons. Model es ima es and Wald s a is ics (Z) wi h p- alues (** <0.01,*<0.05, o <0.1) a e shown. doi:10.1371/jou nal.pone.0157382. 001 Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 8/18 Tall Fescue Endophy es in Eu ope and USA PLOS ONE | DOI:10.1371/jou nal.pone.0157382 June 10, 2016 9/18 ully exploi he longe a o able g owing season in lowe la i udes, he op imal iming o g ow h, ep oduc ion and adap a ions ela ed o win e ha diness such as esou ce alloca ion o s o age o gans a e mo e c i ical o plan i ness in highe la i udes whe e he a o able g ow- ing season is sho e [4]. Ou esul s ha e b oade impo ance o unde s anding he expansion po en ial o species ac oss la i udes. Ou indings sugges ha all escue in asions om highe la i udes o lowe la i- udes a e mo e success ul han he e e se. We p opose ha adap a ions o seasonal changes in pho ope iod in combina ion wi h cold win e s play he key ole causing a al mis iming in he phenology o all escues ansplan ed o highe la i udes. To e u e o suppo his hypo hesis will be le o he u u e s udies explici ly es ing he phenological e en s o all escues in ecip o- cal ansplan a ion expe imen s ac oss la i udes. In pa icula , hese s udies should ake in o accoun ha in na u al popula ions, selec i e o ces a e mo e a iable and can ope a e simul a- neously on se e al ai s, o plas ici y in ai s, o he ungus, hos o hos – ungus uni . In con as o dec eased i ness o he KY-31 plan s in highe la i udes, ou esul s showed ha all no he n o igin all escue ge mplasms, ega dless o hei endophy e in ec ion s a us, pe o med well when ansplan ed o lowe la i udes. This sugges s high po en ial o Eu asian ge mplams in ag i- cul u e in he US, and should be aken in o accoun in all escue b eeding p og ammes. Suppo ing In o ma ion S1 Da a. Biomass and numbe o lowe heads o he expe imen al plan s. (XLS) S1 Fig. G ow h and ep oduc ion o all escue. Biomass (x±S.E.) and numbe o lowe heads (x±S.E.) o wild o igin (A = island o Åland, G = Island o Go land, S = coas al Sweden) and cul i a (Re u and KY-31) plan s ea ed wi h wa e (W), nu ien s (N) o hei combina ion (WN). C = con ol wi h no wa e o nu ien applica ions. (PDF) S2 Fig. Seasonal changes in day leng h a he s udy si es. (PDF) S3 Fig. The expe imen al ield a he Uni e si y o Ken ucky expe imen al a m in Eden Shale, Ken ucky, USA. (JPG) S4 Fig. The expe imen al ield a Tu ku Bo anical Ga den, Uni e si y o Tu ku, Finland. (JPG) S1 Table. Mon hly ain all and mean empe a u e a he s udy si es in Ken ucky and Fin- land. (PDF) Acknowledgmen s We hank Ma ilyn Fae h, Sini Isola, Anna Suu onen, Elina Vainio, Minna Jokela, he s a a Tu ku Uni e si y Bo anical Ga den and nume ous o he people who ha e helped a di e en s ages o he esea ch. 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