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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
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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.
Open Access This a icle is dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea-
i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided you gi e
app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a
link o he C ea i e Commons license, and indica e i changes we e
made.
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