Endophytic Epichloe species and their grass hosts: from evolution to applications

1 3 Plan Mol Biol (2016) 90:665–675 DOI 10.1007/s11103-015-0399-6 Endophy ic Epichloë species and hei g ass hos s: om e olu ion o applica ions Ka i Saikkonen1 · Ca olyn A. Young2 · Ma jo Helande 1,3 · Ch is ophe L. Scha dl4 Recei ed: 4 Sep embe 2015 / Accep ed: 26 Oc obe 2015 / Published online: 5 No embe 2015 © The Au ho (s) 2015. This a icle is published wi h open access a Sp inge link.com Keywo ds Fungal endophy es · G ass · Gene ic a ia ion · Alkaloids · Coe olu ion In oduc ion Specializa ion and coe olu ion ha e aken he cen e s age o discussion in e olu iona y biology since Da win empha- sized in O igin o Species how species di e si y and in e - ac ions oge he shape he e olu ion o li e om indi iduals o communi ies (Da win 1859; Thompson 1994). Now we know ha i ually all species e ol e in in e ac ions wi h o he species, in e ac i e species o en ecip ocally a ec each o he ’s e olu ion, and ecip ocal changes in coe ol - ing species o en equi e and/o p oduce specializa ion. Thus, he majo i y o e olu ion undamen ally inco po a es he elemen s o coe olu iona y p ocesses, and specializa- ion commonly plays a ole, especially in igh ly linked spe- cies in e ac ions such as symbio ic mic obial in e ac ions. In e ac ions be ween endophy ic Epichloë species and hei hos g asses p o ide a unique model o ecologis s and e olu iona y biologis s in e es ed in specializa ion in coe ol ing species in e ac ions. By de ini ion, ungal endo- phy es li e in e nally and asymp oma ically wi hin o gans o hei hos plan (Wilson 1995). These asymp oma ic un- gal in ec ions a e ubiqui ous, abundan and axonomically di e se esiden s in all e es ial plan s (Saikkonen e al. 1998; Rod iguez e al. 2009). The majo i y o endophy es a e la en pa hogens o do man sap ophy es and o he ungal “hi ch-hike s” lu king wi hin he plan issues wi h- ou causing isible symp oms (Wilson 1995; A nold e al. 2000; Saikkonen e al. 2004a, b; Saikkonen 2007; Rod i- guez e al. 2009; Pa ida-Ma inez and Heil 2011; Zabal- gogeazcoa e al. 2013). In con as o many o he ungal axa ha ing asymp oma ic endophy ic pe iods in hei li e Abs ac The closely linked i ness o he Epichloë sym- bion and he hos g ass is p esumed o align he coe olu- ion o he species owa ds specializa ion and mu ually ben- e icial coope a ion. Ecological obse a ions demons a ing ha Epichloë-g ass symbioses can modula e g assland ecosys ems ia bo h abo e- and belowg ound ecosys em p ocesses suppo his. In many cases he de ec ed ecologi- cal impo ance o Epichloë species is di ec ly o indi ec ly linked o de ensi e mu ualism a ibu able o alkaloids o ungal-o igin. Now, mode n gene ic and molecula ech- niques enable he p ecise s udies on e olu iona y o igin o endophy ic Epichloë species, hei coe olu ion wi h hos g asses and iden i ica ion he gene ic a ia ion ha explains pheno ypic di e si y in ecologically ele an cha ac e is ics o Epichloë-g ass associa ions. He e we b ie ly e iew he mos ecen indings in hese a eas o esea ch using he p esen knowledge o he gene ic a ia ion ha explains he biosyn he ic pa hways d i ing he di e si y o alkaloids p oduced by he endophy e. These indings unde sco e he impo ance o gene ic in e play be ween he ungus and he hos in shaping hei coe olu ion and ecological ole in bo h na u al g ass ecosys ems, and in he ag icul u al a ena. * Ka i Saikkonen ka i.saikkonen@luke. i 1 Managemen and P oduc ion o Renewable Resou ces, Na u al Resou ces Ins i u e Finland (Luke), I äinen Pi käka u 3, 20520 Tu ku, Finland 2 The Samuel Robe s Noble Founda ion, A dmo e, OK 73401, USA 3 Sec ion o Ecology, Depa men o Biology, Uni e si y o Tu ku, 20014 Tu ku, Finland 4 Depa men o Plan Pa hology, Uni e si y o Ken ucky, Lexing on, KY 40546-0312, USA 666 Plan Mol Biol (2016) 90:665–675 1 3 cycles, he endophy ic Epichloë species (Leuch mann e al. 2014) ha a e symbio ic wi h cool season g asses o m sys emic and li e-long in ec ions wi hin hei hos s. This ex ension o la ency seen wi h Epichloë species is associ- a ed wi h educ ion o i ulence, adap a ions and special- iza ion ha can p omo e i ness bene i s o he hos g ass (Scha dl 1996; Ko e and Clay 1998; Saikkonen e al. 1998, 2004b, 2006, 2010a; Spa a o a e al. 2007). The symbiosis be ween Epichloë species and g asses is highly in eg a ed in ol ing he ecip ocal use and manipu- la ion o mo phology, physiology, and li e cycle and his o y ai s o he pa ne s o inc ease he i ness o he symbio a. Fi s , he ungal hypha g ows h oughou he abo e-g ound issues o he hos g ass including in lo escences. I emains es ic ed o he in e cellula spaces. Such an in ima e ela- ionship equi es adap a ions allowing he ungus o access he hos plan in e io , pe haps supp essing he ecogni ion and de ense esponses ha no mally hal he es ablishmen o ha m ul ungal in ec ions in he hos plan (Hamil on e al. 2012; Saikkonen e al. 2013a). The associa ed mech- anisms a e poo ly unde s ood bu he oxida i e balance is sugges ed o play a ole (Hamil on e al. 2012). Second, he i ness o he pa ne s is igh ly linked, which should a o he e olu ion o in e ac ion owa d educed an agonism and inc eased pa ne ideli y (Thompson 1994; Saikkonen e al. 2002). Epichloë species a e obliga e associa es o g asses sub- sis ing en i ely on he hos g ass. In addi ion o nu ien acquisi ion, g ass ep oduc ion p o ides a dis ibu ion a e- nue o he Epichloë species which a e e ically ansmi - ed in seeds om plan o i s o sp ing. Fo s ic ly asexual Epichloë species e ical ansmission is he only desc ibed means o dis ibu ion, whils pleio opic Epichloë species a e capable o bo h e ical and ho izon al ansmission wi h asexual o sexual li e cycles (Michalakis e al. 1992; Scha dl 1996; Saikkonen e al. 1998; Tadych e al. 2012). A he o he end o he con inuum, uly sexual Epichloë species a e ho izon ally ansmi ed by ascospo es. Thus, he dis ibu ion o Epichloë species is la gely de e mined by he i ness o he hos pa icula ly in he case o s ic ly asexual Epichloë species (bu see Saikkonen e al. 2002). In exchange o hos ing he endophy e, he hos g ass can ecei e bene i s such as compe i i e supe io i y com- pa ed o unin ec ed coun e pa s in a popula ion h ough inc eased g ow h and ep oduc ion, as well as esis ance o a ious abio ic and bio ic s esses such as d ough , lood- ing, pa hogens and he bi o es (Clay 1988, 2009; Saik- konen e al. 2006, 2010a; Song e al. 2015). Consequen ly, Epichloë species ha e he po en ial o ma kedly a ec hos i ness, exe s ong selec i e p essu e on g ass hos ai s, and modula e g assland ecosys ems (Clay and Holah 1999; Saikkonen 2000; Clay e al. 2004; Rudge s e al. 2004, 2007; Saikkonen e al. 2013a). Simila ly o o he biological in e ac ions based on mu ual exploi a ion, bene i s o Epichloë species and hei hos g asses a e a ely symme ic. Thus, he symbiosis can ange om an agonis ic o mu ualis ic, and con lic ing selec ion o ces a e likely o des abilize hem. Fo exam- ple, when pleio opic and an agonis ic Epichloë species en e hei sexual li e cycle hey p oduce ex e nal s o- ma a su ounding some o all hos in lo escences elimina - ing seed p oduc ion. The bene i s om endophy es appea o be dependen on he ungal and hos geno ype, and on en i onmen al condi ions. Acco dingly, he symbioses a e commonly ega ded ei he as commensal o mu ualis ic. The majo des abilizing o ces in he symbiosis a e asym- me y in dependence and gene ic compa ibili y. Accumu- la ing e idence has e ealed ha he g ass does no neces- sa ily depend on he ungus in some en i onmen s, many Epichloë s ains a e hos species speci ic and gene ic mis- ma ch be ween hos and symbion can limi he endophy e- g ass combina ions (Saikkonen e al. 2004b, 2006, 2010b; Gundel e al. 2010, 2012, 2013). In his pape we i s dissec ecen esea ch ad ances and li e a u e on endophy ic Epichloë species, co e ing hei e olu iona y o igin and axonomical aspec s, unc- ional gene ics, and coe olu ion wi h hos g asses, and hen examine hei ecological oles and po en ial in no el solu- ions o sus ainable ag icul u e. Accumula ing indings ha e e ealed ha Epichloë species can ep og am hos me abolism, and modula e pho osyn hesis, signaling and chemical c oss- alk be ween he pa ne s (Hui u e al. 2014; Ea on e al. 2010, 2015; Dupon e al. 2015) and hus, di ec ly p omo e he g ow h, ep oduc ion and compe i i e abili y o he hos g ass (Clay and Holah 1999; Rudge s e al. 2004, 2007; Saikkonen e al. 2013b). Howe e , he e we ocus on unc ional gene ics d i ing alkaloid p oduc ion because de ense agains he bi o es is sugges ed o be he p ima y d i ing selec i e o ce behind he mu ualism (Clay 2009; Saikkonen e al. 2010a). Specia ion o endophy ic Epichloë species and hei cophylogeny wi h g asses To unde s and he e olu ion o Epichloë species, i is nec- essa y o conside he ela ionships o symbion and hos - plan li e cycles—bo h sexual and asexual—and how hose ela e in u n o ho izon al e sus e ical ansmission, and o haploid e sus polyploid genomes. The Epichloë species, as cu en ly ecognized by mos , a e sys emic symbion s (o pa asi es) in he ae ial pa s o hos plan s in he C3 “cool season” g asses (Poaceae sub amily Pooideae), and ei he a e choke pa hogens o a e ela ed o choke pa hogens (Whi e 1993; Leuch mann e al. 2014). Mos Epichloë species can benignly colonize 667Plan Mol Biol (2016) 90:665–675 1 3 de eloping lo e s and seeds, acili a ing e icien e ical ansmission (Siegel e al. 1984; Tin je e al. 2008). Ho i- zon al ansmission o some Epichloë species can occu ei he ia asexual o sexual spo es (Saikkonen e al. 2004a, b; Tadych e al. 2012). The choke pa hogens can ui on hei hos s, o ming a spo ogenous s oma on he lag-lea shea h and hal ing ma u a ion o he sub ending in lo es- cence (“choke” o “ca ail” disease) (Whi e 1997). The s oma p oduces spe ma ia and ichogynes ( emale ecep- i e hyphae) and a ac s emale Bo anophila sp. lies as “pollina o s” ha ans e spe ma ia. This esul s in c oss- e iliza ion o he A and B ma ing ypes (MTs), which a e de e mined by he MTA and MTB idiomo phs (al e na- i e genes o gene clus e s) a he MT locus (Scha dl and Sco 2012; Scha dl e al. 2014). The ensuing sexual s age gene a es haploid spo es (“ascospo es”) ha can media e ho izon al ansmission o de eloping seeds (Chung and Scha dl 1997a) o g owing plan s (Meije and Leuch mann 1999). In some hos s, sexual Epichloë species a e obse ed only o ansmi ho izon ally, bu in mos he e can be a mix u e o choked ille s and asymp oma ic ille s, wi h he la e bea ing he endophy e in he seeds (Sampson 1933; Whi e 1994; Scha dl 2001). Since he e is no gene ic di e ence in he ungus associa ed wi h choked e sus asymp oma ic ille s on an indi idual plan , i seems likely ha his dual- i y o ep oduc i e p ocesses has an epigene ic basis. Mos Epichloë species a e incapable o ui ing on hei hos s, and a e he e o e asexual and e ically ansmi ed. Some o hese can s ill o m spa se hyphal ne s ha p o- duce some conidia (Whi e e al. 1996), and heo e ically could ansmi ho izon ally as well, bu e ical ans- mission seems by a he dominan p ocess o asexual Epichloë species. (Whi e e al. 1991; Moon e al. 2000, 2002; Chen e al. 2015) The majo i y o asexual Epichloë species a e diploid o iploid in e speci ic hyb ids pos- sessing mos o all o he genomes o wo o h ee ances- al haploids, espec i ely. Such polyploid, asexual hyb ids seem unusual among ungi, hough well documen ed in he Ve icillium dahliae species complex (Inde bi zin e al. 2011), and a common cha ac e is ic o pa henogenic liz- a ds, ish, amphibians (Boga e al. 2007; Lampe and Scha l 2010; Cha ney 2012) and nema odes (Lun 2008). The lack o a ege a i e incompa ibili y sys em in Epichloë species (Chung and Scha dl 1997b), and p esumably selec- ion a o ing some hyb ids o e ances al haploids can accoun o he abundance o hyb id Epichloë species in na u e (Fae h and Saa i 2012). In es iga ion o possible cophylogeny o haploid Epichloë species wi h hei pooid g ass hos s would sug- ges when his symbio ic sys em i s eme ged. The gene ic analysis o Epichloë species and g asses indica ed signi i- can hos -endophy e co-di e gence (Scha dl e al. 2008). Since hen, an explosion o genome sequences o Epichloë species and ela ed Cla icipi aceae has allowed mo e de ailed phylogene ic analysis (Leuch mann e al. 2014; Scha dl e al. 2014; Chen e al. 2015). S ikingly, he deep- es spli iden i ied is o a clade o wo species symbio ic wi h Achna he um species (Fig. 1) whose ibe (S ipeae) g oups in a clade ha spli s ea ly om mos o he o he pooid ibes om which Epichloë species ha e been sam- pled (GPWG 2001). Almos as basal is he b anch o E. glyce iae, which is associa ed wi h ano he ea ly di e ging ibe (Meliceae). This con as s wi h a clade in mos house- keeping gene ees ha includes E. b omicola and E. elymi, o which he o me is ound in membe s o sis e ibes Ho deeae (=T i iceae) and B omeae, and he la e is ound jus in Ho deeae. Simila ly, ano he clade encompasses se - e al Epichloë species ha a e ound only in Poeae; namely, E. ama illans, E. baconii, E. es ucae, E. mollis and E. s o- ma olonga. Cophylogeny is no consis en ly indica ed o all species and clades (pa icula ly no o he b oad hos ange species Epichloë yphina), bu e idence o a signi i- can endency o co-di e gence sugges s ha he o igin o genus Epichloë may ha e been close in ime o he o igin o he highly speciose g ass sub amily, Pooideae (Scha dl e al. 2008; Bouchenak-Khelladi e al. 2010; Amb ose e al. 2014). Mos o bu no all asexual Epichloë species a e in e - speci ic hyb ids (Moon e al. 2004; Cha l on e al. 2012, 2014; Iannone e al. 2012; Obe ho e and Leuch mann 2012; McCa go e al. 2014; Scha dl e al. 2012). In hos s o egions whe e a single hyb id Epichloë species domina es, he implica ion is ha he p oduc o ha hyb idiza ion was an endophy e ha so enhanced i s hos ’s i ness ha i sp ead h ough much o all o i s hos ’s ange. An exam- ple is Epichloë uncina a (E. b omicola × E. yphina subsp. poae), which is ound only in he g ass Lolium p a ense (=Schedono us p a ensis = Fes uca p a ensis; meadow escue), and h oughou he geog aphical ange o i s hos (Ekanayake e al. 2012; Ka imi e al. 2012). Only one iso- la e om his g ass has been iden i ied as a dis inc Epichloë species, E. siegelii = E. b omicola × E. es ucae (C a en e al. 2001). Likewise, he h ee-pa hyb id, Epichloë coe- nophiala, domina es popula ions o Lolium a undinaceum (=Schedono us a undinaceus = Fes uca a undinacea; all escue) in no he n Eu ope, cen al Asia (Ekanayake e al. 2012) and I an (Ka imi e al. 2012). On ano he con inen , E. emblade ae has been ound in nume ous na i e g asses h oughou A gen ina (Iannone e al. 2012), including wo species wi h a second hyb id endophy e; namely, B omus aule icus, which can also hos Epichloë pampeana (also E. es ucae × yphina subsp. poae) (Iannone e al. 2009), and Phleum alpinum, which can also hos Epichloë cab alii (E. yphina subsp. poae × a ela i e o E. baconii) (McCa go e al. 2014). 668 Plan Mol Biol (2016) 90:665–675 1 3 Al hough an inc easing numbe o s udies sugges con- ex dependency o i ness bene i s o he hos g ass om hyb id endophy es depending on e.g. en i onmen al condi- ions and cascading ophic in e ac ions, hyb id endophy es appea o inc ease hos adap abili y especially o ex eme en i onmen s (Hamil on e al. 2009, 2010; Fae h and Saa i 2012; Obe ho e e al. 2014; Saa i e al. 2014). One possible eason o in e speci ic hyb ids o p o ide excep ional i ness con ibu ions o hos g asses is he p o- duc ion o an i-he bi o e alkaloids. Bo h E. coenophiala and E. uncina a, and also E. siegelii, p oduce e y high le els o loline alkaloids, which p o ide b oad-spec um p o ec ion om insec s (Scha dl e al. 2007). Mos E. coe- nophiala s ains also p oduce e go alkaloids a le els ha de e g azing by li es ock, as well as he insec eeding de e en , pe amine (Ch is ensen e al. 1993; Bush e al. 1997). Many o he endophy es in A gen ina ha e genes o indole-di e pene biosyn hesis, and his i s wi h symp- oms su e ed by li es ock ha inges Poa huecu, which commonly hos s E. emblade ae (Cab al e al. 1999). Such poisonings a e some imes epo ed o be a al o he ani- mal. The o he A gen ine endophy es, E. pampeana and E. Fig. 1 Phylogeny o TubB coding sequences (cds) o Epichloë spe- cies and ela ed species. Gene coding sequences we e iden i ied by manual anno a ion o sequenced genomes. The ee was in e ed by maximum likelihood sea ch using PhyML wi hou GBlocks cu a- ion. The ee was oo ed wi h Fusa ium g aminea um PH-1 as he ou g oup, and numbe s on b anches indica e ALR suppo . Alkaloid biosyn hesis gene clus e s and ma ing ypes a e indica ed a e each s ain designa ion, wi h symbols de ined in he legends benea h he phylog am. Alkaloid gene clus e s a e o e go alkaloids (EAS), indole-di e penes (IDT) and lolines (LOL); and he mul i-domain pe amine syn he ase gene (pe A) and a ela ed allele (pe A-∆R*) a e also indica ed 669Plan Mol Biol (2016) 90:665–675 1 3 cab alii, also p oduce lolines (McCa go e al. 2014). The equency o alkaloid genes appea ing in hyb ids is e y high, sugges ing ha he alkaloids cons i u e a signi ican componen o he i ness enhancemen ha is he basis o selec ion o he hyb id endophy es, as discussed in he ol- lowing sec ion. Gene ic di e si y o Epichloë species and alkaloid p o ile o symbio a The bioac i e alkaloids, e go alkaloids, indole-di e penes, lolines and pe amine, can be p oduced by Epichloë species and likely p o ide selec i e ad an ages o he hos species he endophy es inhabi . The lolines a e s ongly insec icidal and pe amine ac s as an insec eeding de e en (Siegel e al. 1990; Riedell e al. 1991). The e go alkaloids and indole-di e penes a e mos well known o hei oxici y o g azing li es ock in he o m o escue oxicosis and yeg ass s agge s, espec i ely, bu can also exhibi an i- insec ac i i y. To unde s and alkaloid p oduc ion ini ial esea ch ocused on iden i ica ion o pa hway end p oduc s o each alkaloid class. Gene ics and molecula biology we e used o iden i y genes encoding each pa hway s ep and ecom- binan echnology was used o dissec he biosyn he ic pa hways by gene knockou s, RNAi and he e ologous gene exp ession (Panaccione e al. 2001; Spie ing e al. 2002, 2005, 2008; Wang e al. 2004; Tanaka e al. 2005; Young e al. 2005, 2006; Saikia e al. 2012; Pan e al. 2014a, b). Apa om he pe A gene ha encodes pe amine syn- he ase, he o he alkaloid loci (EAS o e go alkaloids, IDT/LTM o indole-di e penes and LOL o lolines) a e gene clus e s ha a e o en complica ed by he p es- ence o AT- ich epe i i e sequences. Genome sequencing has enligh ened us on he ex ensi e gene ic di e si y o Epichloë species wi h espec o he known alkaloids and also p o ides in o ma ion on o he biosyn he ic gene clus- e s, o many o which he p oduc s a e ye o be eluci- da ed (Scha dl e al. 2013a, b, 2014). Compa ison o gene and genome sequences om spe- cies wi h di e ing alkaloid p o iles has p o ided insigh in o he gene ic a ia ion ha explains endophy e chemo- ypic di e si y (Scha dl e al. 2013a, b, 2014; Be y e al. 2015). S ains o Epichloë species ha a e unable o p o- duce a speci ic alkaloid class a e ypically de oid o genes encoding key pa hway s eps. In many cases he whole gene ic locus is absen , bu some imes emnan genes, pseudogenes o gene agmen s om he locus can s ill be iden i ied wi hin he genome. Chemo ypic di e si y wi hin a gi en pa hway can also be iden i ied. Fo example, he chemo ypic di e ence be ween wo E. canadensis s ains symbio ic wi h Elymus canadensis ha a y wi hin e go alkaloid (chanocla ine s. e go aline) and loline alkaloid (1-ace amidopy olizidine s. N-ace ylno loline) pa hway end p oduc s a e explained by a ia ion o he genes ha a e p esen (Cha l on e al. 2012; Scha dl e al. 2013b; Pan e al. 2014a, b). The E. canadensis isola e CWR5 has a unc ional EAS locus con aining all 11 EAS genes enabling p oduc ion o e go aline and a LOL locus o p oduc ion o N-ace ylno loline ha lacks unc ional copies o lolP, lolM and lolN. (No e ha isola es capable o p oducing N- o mylloline would ha e unc ional copies o lolP, lolM and lolN). The E. canadensis isola e CWR34 lacks mos EAS genes con aining only unc ional copies o dmaW, easF, easC and easE encoding he s eps o chanocla- ine. The CWR34 LOL locus is simila o CWR5 excep a small dele ion in lolO ende s he gene non- unc ional so he pa hway s ops ea lie a 1-ace amidopy olizidine. Many o he examples exis whe eby he gene ic a ia ion be ween s ains can explain di e ences in alkaloid chemo- ypes (Cha l on e al. 2014; Takach and Young 2014; Young e al. 2014, 2015; Be y e al. 2015). In a e cases a whole gene clus e is p esen and con ains no appa en dele e ious mu a ions ye he co esponding alkaloid is no p oduced. I appea s ha hese clus e s a e silen and gene exp ession is below a h eshold le el o unc ionali y (Scha dl e al. 2013b; Cha l on e al. 2014). Jus as gene con en can a y o an alkaloid locus, so can he gene a angemen wi hin a locus. Many o he clus e ea angemen s ha e likely occu ed due o epe i- i e sequences wi hin each locus. In e es ingly he loci o e go alkaloids and indole-di e penes a e loca ed a a sub- e minal egion o he ch omosome, al hough he alkaloid genes loca ed nea es he elome e can a y. The EAS clus- e s ep esen he g ea es a ia ion o clus e o ganiza ion ac oss Epichloë species wi h a leas i e di e en genes posi ioned nea es he elome e dependen on species o s ain (Scha dl e al. 2013a; Young e al. 2015). The polyploid na u e o he in e speci ic hyb id genome means ha one o all ances o s can con ibu e alkaloid genes, which can allow o py amiding o alkaloid classes (Fig. 2). Con ibu ing ances al species can be iden i- ied h ough phylogene ic analysis o he alkaloid genes and mos o en hese a e consis en wi h he species ee (Scha dl e al. 2013b, Cha l on e al. 2014, Be y e al. 2015). The alkaloid gene con ibu ions in hyb id species a e equen ly ound in he ex an nonhyb id species. The chanocla ine geno ype, EASCC, is p esen in he hyb ids E. canadensis (hyb id o E. ama illans × E. elymi) and E. unkii (E. elymi × E. es ucae) and is con ibu ed by E. elymi. The e a e o he examples whe e he alkaloid gene con ibu ion has only been ound in he hyb id species and no in he ex an ances o . The LOL gene o igin in E. coe- nophiala is E. yphina subsp. poae, bu LOL genes a e ye o be iden i ied in his species (Ku il e al. 2007). 670 Plan Mol Biol (2016) 90:665–675 1 3 In addi ion o alkaloid di e si y, some hos species a e able o o m a symbio ic associa ion wi h di e en endophy e species. Tall escue, B omus lae ipes, Elymus canadensis, Ho delymus eu opaeus, Achna he um obus- um and Achna he um ineb ians can independen ly hos mo e han one Epichloë species bu his associa ion is s ill limi ed o only one endophy e s ain pe indi idual plan (Ch is ensen e al. 1993; Obe ho e and Leuch mann 2012; Scha dl e al. 2013b; Cha l on e al. 2014; Takach and Young 2014; Chen e al. 2015; Shymano ich e al. 2015). The symbion a ia ion can be u he expanded due o alkaloid chemo ypic a ia ion wi hin a single species. Con- e sely, some imes he same Epichloë species can be ound in di e se hos species. Fo example E. emblade ae is ec- ognized as a symbion o Poa huecu, Fes uca a izonica, Fes- uca a gen ina, and B omus aule icus (Cab al e al. 1999; Moon e al. 2004; Iannone e al. 2009) bu i is unknown i chemo ypic a ia ion exis s ac oss his endophy e species. The ull ex en o alkaloid di e si y associa ed wi h Epichloë species is only now being ealized as ou abil- i y o gene ically e alua e he endophy e di ec ly wi hin he plan has imp o ed (Cha l on e al. 2014; Takach and Young 2014; Young e al. 2014, 2015; Chen e al. 2015; Shymano ich e al. 2015). How his endophy e di e si y is ansla ed o hos i ness enhancemen needs o be u he explo ed. Ecological consequences o gene ics in na u e and man‑made en i onmen s The g owing li e a u e illus a es he impo ance o gene - ics o symbio ic Epichloë species and hei hos g asses in bo h e olu iona y and ecological ime-scales. Phyloge- ne ic analyses sugges he co-o igin o genus Epichloë and he g ass amily Pooideae (Scha dl e al. 2008) explaining high p e alence o Epichloë species in his pa icula g ass amily. Co-phylogeny is no consis en o all species and clades bu in co-di e ged phylogene ic b anches hyb idi- za ion is commonly de ec ed (Moon e al. 2004; Cha l on e al. 2012; Iannone e al. 2012; Obe ho e and Leuch - mann 2012; McCa go e al. 2014). Gene ic compa ibili y be ween he ungal s ain and he hos lineage appea s o play signi ican ole in es ablishmen o endophy e-g ass combina ions, and ansgene a ional ma e nal e ec s can a ec he gene ic s uc u e o a hos popula ion (Saikkonen e al. 2010b). Gene ic a ia ion be ween Epichloë s ains explains di e ences in alkaloid chemo ypes (Cha l on e al. 2014; Takach and Young 2014; Young e al. 2014, 2015; Be y e al. 2015) and he equency o alkaloid genes is high in hyb ids allowing o py amiding o alkaloid classes. Thus, hyb idiza ion can esul in signi ican i ness enhancemen and selec i e ad an age o hyb id endophy es. Howe e , gene ic di e ences among ungal lineages ail o Fig. 2 Ploidy o hyb id species and closes ances o con ibu ing alkaloid genes. The closes ances o s a e indica ed as E. ama illans (Eam), E. baconii (Eba), E. b omicola (Ebo), E. elymi (Eel), E. es u- cae (E e), E. yphina (E y) and E. yphina subsp. poae (E p). Alkaloid gene clus e s a e o e go alkaloids (EAS), indole-di e penes (IDT) and lolines (LOL); and he mul i-domain pe amine syn he ase gene (pe A) and a ela ed allele (pe A-∆R*) a e also indica ed. Unnamed Epichloë axa a e abb e ia ed by hos , B. lae ipes Taxonomic G oup (BlaTG-#) and F. a undinacea Taxonomic G oup (FaTG-#) 671Plan Mol Biol (2016) 90:665–675 1 3 explain o example, he duali y o ep oduc i e sexual and asexual s a egies o he ungi. These obse a ions sugges ha specializa ion and gene ic in e play be ween he endo- phy e and he hos g ass can la gely explain pheno ypic a ia ion in he symbio um and i s ecological consequences bu also sugges s ha o he mechanisms such as pheno ypic plas ici y and epigene ic modi ica ions in gene exp ession and unc ion a e likely o play a signi ican ole in ecologi- cally ele an ai s o he Epichloë-g ass symbiosis. In na u e, endophy ic Epichloë species can a ec he hos g ow h and ep oduc ion, he s uc u e o g assland communi ies and ophic in e ac ions, and he eby adap i e adia ion o Epichloë species and hei hos g asses (see e.g. Clay and Scha dl 2002; Clay e al. 2004; Rudge s e al. 2004, 2007; Saikkonen e al. 2004a, 2006, 2010a; Rod i- guez e al. 2009). Because g asses domina e app oxima ely 40 % o he Ea h’s su ace, Epichloë species a e likely o ha e signi ican ecosys em consequences as well. The po en ial applica ions a e ela ed o success ul g ass p oduc ion managemen in he changing clima e. Fo example, economical alue o sys emic g ass-endo- phy es ela ed o o age quali y and biocon ol has al eady been widely ecognized in ag icul u e and u g ass indus y in he USA and New Zealand (Ho eland 1993; Gundel e al. 2013; Johnson e al. 2013). Economic losses caused by poo animal pe o mance eeding on endophy e in ec ed o age o all escue and pe ennial yeg ass in he Uni ed S a es only ha e been es ima ed a $600 mil- lion annually (Ho eland 1993). On he o he hand, un- gal s ains which do no p oduce myco oxins ha m ul o ca le bu inc ease biomass p oduc ion, seed p oduc ion and ge mina ion, s ess ole ance (e.g. d ough , looding, empe a u e, and pes , pa hogen and weed in asions), sili- con, seconda y me aboli e o nu ien con en should be aken in o accoun when aiming o inc ease o age p o- duc i i y when in oduced o o age cul i a s (Clay and Scha dl 2002; Leh onen e al. 2006; Saikkonen e al. 2013b; Vázquez-de-Aldana e al. 2013; Hui u e al. 2014; Song e al. 2015). One o he mos success ul comme - cial example o such an animal-sa e non- oxic endophy e is ‘‘MaxQ’’ (E. coenophiala) in he all escue a ie y “Jesup” (Johnson e al. 2013). Examples o comme cially success ul no el endophy es p o iding bio-p o ec i e p ope ies o he hos plan agains insec pes s a e e.g. “AR1, AR5, AR37 and NEA2” endophy e s ains which ha e been selec ed and ans e ed o pe ennial yeg ass cul i a s (Johnson e al. 2013). Di e en endophy e s ains, howe e , exhibi ema kable a ia ion in alkaloid ypes, and le els o alkaloids a e con ex dependen (Bony e al. 2001; Johnson e al. 2013). The posi i e e ec s o endophy es appea o be mo e p onounced in nu ien - ich en i onmen s. Recen e idence sugges s ha also wa m- ing and d ough s ess can a ec he alkaloid p oduc ion o endophy es (Hill e al. 1996; B osi e al. 2009; Com- pan e al. 2010) sugges ing ha de ensi e mu ualism should be aken in o accoun in g ass p oduc ion manage- men in he changing clima e. The associa ed economic and ood sa e y p o i s o using hese endophy e imp o ed g ass cul i a s include lowe in es men s in chemical pes con ol when using na u al biocon ol and consume s a oid emnan s o chemical pes icides in he c op, mea and milk. Cu en bio echnological knowledge allows us o u i- lize endophy ic Epichloë species in ag ibusiness (Gundel e al. 2013; Johnson e al. 2013). Endophy ic ungi can be ou inely elimina ed om hos plan seeds by hea ea - men s o ungicides and new s ains in oduced in o he unin ec ed plan s by inocula ing he hyphae in o he plan issue. Fu he mo e, ecen disco e ies in genome map- ping echniques allow iden i ica ion and loca ion o genes ha encode he in o ma ion ha ha e ecological impo - ance. In addi ion o enhanced myco oxin p oduc ion we a e only beginning o unde s and gene ic bases o o he adap i e ungal and g ass ai s. This knowledge and he ools o con empo a y gene ics widen he possibili ies o plan b eeding om u iliza ion o selec ed endophy e- plan manipula ions o he ans e o gene(s) om he ungus o g asses o o he c op plan s such as ce eals. Tho ough unde s anding o mechanisms unde lying a i- a ion, he i abili y and s abili y o cul i a ai s a e, how- e e , equi ed o unde s and esponses o g ass cul i a s o en i onmen al change and hei success ul use in di - e en en i onmen s. Conclusions and u u e pe spec i es The impo ance o endophy ic Epichloë species o ocal eco- sys em unc ions d i ing bo h below- and abo eg ound ood webs is well ecognized and accep ed (Omacini e al. 2001; Clay and Scha dl 2002; Clay e al. 2004; Rudge s e al. 2004; Saikkonen e al. 2006, 2010a, 2013a, b, 2015; Rudg- e s e al. 2007; Omacini e al. 2012). Recen phylogene ic and molecula analyses coupled wi h accumula ing ecologi- cal app oaches ha e p o ided insigh s in o he coe olu ion o Epichloë-g ass symbiosis and how gene ic in e play be ween he pa ne s can ha e g ea epe cussions also in a ecologi- cal ime-scale. Rep oduc ion and ansmission mode ( e i- cal s. ho izon al) o Epichloë species as well as a chi ec u e and li espan o he hos g ass a e impo an ac o s ela ed o he epidemiology, gene ic compa ibili y, specializa ion and e olu ion o a i ulence in Epichloë species. Howe e , he gene al ques ions o be sol ed in u u e s udies a e (a) wha is he ela i e impo ance o pheno ypic plas ici y and he i - able (gene ic and/o epigene ic) a ia ion in ecologically el- e an g ass ai s, (b) how selec ion ope a es on he uni a y, 672 Plan Mol Biol (2016) 90:665–675 1 3 modula o supe o ganism le els o Epichloë-g ass associa- ions, (c) how he pheno ypic uni o he symbio um medi- a es plan –plan and ophic in e ac ions in g assland com- muni ies, and (d) species dis ibu ion anges. Un il now he lack o his knowledge has limi ed he use o ull po en ial o endophy ic Epichloë species in sus ainable ag icul u e. Acknowledgmen s This s udy was inancially suppo ed by Finn- ish Academy G an s 137909, 281354 and 292732, USDA-CSREES G an 2009-34457-20125, USDA-CSREES G an 2010-34457- 21269, USDA-NIFA G an 2012-67013-19384, NSF g an EPS- 0814194, Na ional Ins i u es o Heal h G an s R01GM086888 and 2 P20 RR-16481, and he Samuel Robe s Noble Founda ion. 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