Gene ic Compa ibili y De e mines Endophy e-G ass
Combina ions
Ka i Saikkonen
1
*, Piippa R. Wa
¨li
2
, Ma jo Helande
3
1Plan P oduc ion Resea ch, MTT Ag i ood Resea ch Finland, Jokioinen, Finland, 2Depa men o Biology, Uni e si y o Oulu, Oulu, Finland, 3Depa men o Biology,
Uni e si y o Tu ku, Tu ku, Finland
Abs ac
E en highly mu ually bene icial mic obial-plan in e ac ions, such as myco hizal- and hizobial-plan exchanges, in ol e
sel ishness, chea ing and powe -s uggles be ween he pa ne s, which depending on p e ailing selec i e p essu es, lead o
a con inuum o in e ac ions om an agonis ic o mu ualis ic. Using manipula ed g ass-endophy e combina ions in a i e
yea common ga den expe imen , we show ha g ass geno ypes and gene ic misma ches cons ain gene ic combina ions
be ween he e ically ( ia hos seeds) ansmi ed endophy es and he ou -c ossing hos , he eby educing in ec ions in
es ablished g ass popula ions. In ec ions we e los in bo h g ass ille s and seedlings in F
1
and F
2
gene a ions, espec i ely.
Expe imen al plan s we e collec ed as seeds om wo di e en en i onmen s, i.e., meadows and nea by i e banks.
Endophy e- ela ed bene i s o he hos included an inc eased numbe o in lo escences, bu only in meadow plan s and no
un il he las g owing season o he expe imen . Ou esul s illus a e he impo ance o gene ic hos speci ici y and ans-
gene a ional ma e nal e ec s on he gene ic s uc u e o a hos popula ion, which ac as des abilizing o ces in endophy e-
g ass symbioses. We p opose ha (1) gene ic misma ches may ac as a bu e ing mechanism agains highly compe i i e
endophy e-g ass geno ype combina ions h ea ening he biodi e si y o g assland communi ies and (2) hese misma ches
should be acknowledged, pa icula ly in b eeding p og ammes aimed a ha nessing sys emic and he i able endophy es o
imp o e he ag icul u ally aluable cha ac e is ics o cul i a s.
Ci a ion: Saikkonen K, Wa
¨li PR, Helande M (2010) Gene ic Compa ibili y De e mines Endophy e-G ass Combina ions. PLoS ONE 5(6): e11395. doi:10.1371/
jou nal.pone.0011395
Edi o : Dennis Ma inus Hansen, S an o d Uni e si y, Uni ed S a es o Ame ica
Recei ed Ma ch 31, 2010; Accep ed June 10, 2010; Published June 30, 2010
Copy igh : ß2010 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.
Funding: This s udy was unded by he Academy o Finland (P ojec no. 110658, www.aka. i). The unde s had no ole in s udy design, da a collec ion and
analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: ka i.saikkonen@m . i
In oduc ion
Mu ualis ic in e ac ions be ween mic obes and plan s a e
iewed as a ubiqui ous coope a ion con e ing ecip ocal bene i s
o he pa ne s. Howe e , e en seemingly highly mu ualis ic
in e ac ions (e.g. be ween plan s, myco hizal ungi and/o
hizobia) a e inhe en ly uns able, because ecip ocal coope a ion
is based on mu ual exploi a ion and hus cos s and bene i s a e
a ely symme ic o he pa ne s [1–7]. Consequen ly, mic obial-
plan in e ac ions, like any o he biological in e speci ic in e ac ion
[1,2,8–12], in ol e sel ishness, chea ing and powe -s uggles
be ween he pa ne s, hus o ming a con inuum o in e ac ions
om an agonis ic o mu ualis ic [4], wi h an occasional b eakdown
in mu ualism [12].
The symbiosis be ween endophy es and g asses is gene ally
conside ed o be a classic example o mic obe-plan mu ualism
d i ing g assland communi ies [13], as well as hose ood webs
subsis ing upon hem [14,15]. The close link be ween endophy e
i ness and i s hos g ass is p esumed o align he in e es s o bo h
pa ne s owa ds a mu ually bene icial coope a ion [6,7,15,16], a
iew which seems o be suppo ed by empi ical e idence. In his
highly in eg a ed symbiosis, hyphae g ow in e cellula ly and
asymp oma ically h oughou he abo e-g ound issues o he hos
g ass. Th ough g owing in o he de eloping in lo escence and
seeds, he ungus is e ically ansmi ed om ma e nal plan o
o sp ing. E olu iona y e idence o s ic ly asexual Neo yphodium
and sexual Epichloe¨ endophy es sugges s ha such e ical
ansmission is concomi an wi h a educed abili y o con agious
sp eading by asexual o sexual spo es and gene ic hos speci ici y
[16]. Because he i ness and dis ibu ion o a ungus la gely
depends on hos i ness [6], any mu ualis ic coope a ion p o iding
a selec ion ad an age o he hos plan also bene i s he ungus.
Con e sely, ecip ocal bene i s om he ungus o he hos plan ,
such as inc eased g ow h, esis ance o bio ic and abio ic s esses
and enhanced compe i i e abili ies [15], u he suppo he idea
o endophy e-g ass mu ualism [16].
Ne e heless, in mos endophy e-g ass in e ac ions pa ne
bene i s and symbio ic dependence a e asymme ic [6]. Symbiosis
is essen ial o an endophy e because du ing i s sys ema ic g ow h
he ungus subsis s en i ely on and wi hin he hos g ass and
e ical ansmission ia hos seeds is he p ima y mode o ungal
dis ibu ion [16]. By con as , he symbio ic ela ionship emains
only condi ional o he hos plan , as plan i ness does no
necessa ily depend on he ungus [4,6,15]. In ac , in some
en i onmen s symbiosis may e en be maladap i e [17,18]. Fo
example, in endophy e species capable o sexual ep oduc ion, he
p oduc ion o i s ui ing body is cos ly o he hos in e ms o
p e en ed lowe ing [19]. Fu he mo e, in comple ely asexual
endophy e s ains, he adap i e alue o symbiosis o he hos g ass
appea s o a y among ungal s ains, being mo e p onounced in
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nu ien - ich en i onmen s [15], as well as being dependen on
plan -plan in e ac ions in g assland communi ies [13,20] and
ophic in e ac ions in ood webs [16,21,22,23]. Acco dingly, he
in ec ion incidence o g ass species and popula ions appea s o be
highly a iable spa io empo ally [24–28], e lec ing how ungus
and hos alike espond o changing selec ion p essu es, ei he
indi idually o as a pheno ypic uni [6].
He e, we use endophy e manipula ion ials and a i e yea
common ga den expe imen o es he impo ance o gene ic
compa ibili y o endophy e-g ass symbiosis. Gene ic compa ibili y
was examined in h ee ansgene a ional phases om he pa en al
plan gene a ion o hose o he F
1
and F
2
gene a ions; i s a he
ini ial encoun e o he ungus and he g ass, hen in he success o
he e ical ansmission o he ungus o he ege a i e p opagules
( ille s) and o sp ing o he hos g ass. The easoning is ha he
asymme ic dependence o he endophy e and he hos g ass may
lead o (1) hos plan sanc ions agains less bene icial ungal s ains
in p e ailing selec i e p essu es and (2) he loss o he e ically
ansmi ed ungus, which is con inually con on ed wi h new
gene ic combina ions in he ou -c ossing hos popula ion. This is
because he endophy e geno ype emains unchanged in he plan
lineage whils plan geno ypes a e blended h ough ecombina ion
o e ime [6]. This could lead o a gene ic misma ch be ween he
ungus and he hos , hus des abilizing he symbiosis and
cons aining he di e si y o success ul geno ype-geno ype combi-
na ions o he e ically ansmi ed endophy es and he hos
g asses.
Ma e ials and Me hods
S udy sys em
To cap u e he b ead h o a iabili y inhe en in g ass-
endophy e symbioses, as a sui able model we selec ed wild
popula ions o a na i e g ass species, i.e. ed escue (Fes uca ub a
L.) [29], in suba c ic Finland. This species belongs o a la ge and
ubiqui ous g oup o mo phologically simila ine lea ed Fes uca
species. Because o hei high ole ance o a wide ange o bio ic
and abio ic condi ions, escues ha e been enacious in ade s o
e es ial habi a s, colonising e e y con inen on he Ea h in
g ea e abundance and dis ibu ion han any o he g oup o highe
plan s [30]. Fu he mo e, hey a e also o g ea ag onomic
impo ance in ameni y u . Finnish ed escue popula ions a e
in ec ed by he sys emic and e ically ansmi ed Epichloe¨ es ucae
Leuch m., Scha dl & Siegel endophy e [27]. Al hough a
subs an ial numbe o na i e popula ions a e endophy e- ee
[24,27], he p opo ion o in ec ed plan s anges om 4 o 87%
in in ec ed popula ions and is highes in suba c ic a eas [24,27].
Fu he mo e, in ec ion equencies appea o be habi a - ela ed,
being highe in meadows compa ed o i e banks, wi hou gene ic
di e en ia ion in endophy e popula ions among he habi a s [27].
This sugges s ha he selec ion ad an age o he symbiosis a ies
be ween en i onmen s. A single ungal geno ype ( ep esen ing
63.5% o all isola es) domina es he suba c ic endophy e
popula ions. The gene ic di e si y de ec ed appea s o be
un ela ed o ei he he in ec ion equency o habi a [27],
sugges ing ha he ungus is p edominan ly asexual a he edge
o i s no he n dis ibu ion ange in he suba c ic. This supposi ion
is s ongly suppo ed by he ac ha in ou 12 yea s o in ensi e
ieldwo k in ol ing Fes uca ub a in no he nmos Finland, no
endophy ic sexual ui ing bodies ha e e e been de ec ed.
Success o e ical ansmission in na u e
We collec ed ed escue (Fes uca ub a) seeds om 110 wild plan s
(pa en al gene a ion) g owing in ei he meadow (six popula ions; 11,
10, 14, 11, 12 and 10 plan s pe popula ion) o i e bank ( ou
popula ions; 10, 10, 10 and 12 plan s pe popula ion) habi a s in
suba c ic i e alleys in no he nmos Finland in all 2000. The
in ec ion s a us o he p ogenies was i s de e mined by he
mic oscopic examina ion o s ained seeds collec ed in he ield [31],
as well as s ained lea shea hs o he es ablished seedlings [32].
Endophy e manipula ions o es gene ic hos speci ici y
Ou p ima y in en ion was o gene a e F
1
popula ions consis ing
o con olled geno ype-geno ype combina ions o he ungus and
he plan (including na u ally endophy e-in ec ed (E+) and
endophy e- ee (E-) con ols) o explici ly examine o wha ex en
he pheno ypic ai s o hos - ungal uni s a e explained by ungal
associa e and gene ic combina ions be ween he ungus and he
hos . The endophy e manipula ion ials, in ol ing endophy e
emo al om E+seeds by hea ea men , as well as inocula ed E-
seedlings, also allowed us o examine whe he pa ne speci ici y o
Figu e 1. Success o manipula ion ials. Inocula ion success o
ungal endophy e isola es in manipula ion ials wi h 42 na u ally
endophy e- ee ma e nal amilies o Fes uca ub a (A) and 49 Epichloe
¨
es ucae endophy e isola es (B) isola ed om he same popula ions.
Endophy e-in ec ed (E+) and endophy e- ee (E-) seedlings a e shown
as black and whi e ba s.
doi:10.1371/jou nal.pone.0011395.g001
Gene ic Misma ch & Endophy es
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compa ibili y cons ain he di e si y o es ablished endophy e
symbiosis.
Seeds o 87 ma e nal plan s o he pa en al gene a ion,
p oducing ei he s ic ly E+o E- p ogenies, we e assigned o
he endophy e manipula ions. To manipula e in e ac ions be ween
ungal endophy es and g asses, we i s elimina ed he ungus om
some o he E+seeds by hea ea men s (ME-). Using a
modi ica ion o he me hod by Williams e al. [33], in ec ed seeds
we e placed in Eppendo ubes in a wa e incuba o a +54.2uC
o 20 minu es. All seeds we e ge mina ed and hen some o he
na u ally endophy e- ee (E-) seedlings we e in ec ed by inocula -
ing hyphae in o he plan issue (ME+) immedia ely a e he
eme gence o he i s seed lea [34]. The wo week old seedlings
we e po ed in sand and g own in he g eenhouse. The ungal
isola es used in he inocula ions we e isola ed om he same
popula ion as each a ge seedling. The ge mina ion a e o
un ea ed seeds was 51%. Al hough he hea ea men success ully
emo ed he ungus in 99% o he seedlings, he ea men also
deg eased he ge mina ion a e om 51% o 8%. In o al, 3397
seeds ou o 19831 ge mina ed and 2326 seedlings we e subjec ed
o he inocula ion ea men . App oxima ely one hi d (38%) o he
es ablished o iginally endophy e- ee seedlings we e success ully
inocula ed (132 ou o 350 seedlings). Inocula ion dec eased he
su i al o seedlings om 67% o 30%. Finally, gene ic hos
speci ici y be ween he ungus and he hos plan was examined
wi h 42 ma e nal amilies and 49 ungal isola es. The in ec ion
Figu e 2. Loss o in ec ion in ege a i e g ass ille s. P opo ion
(%) o Epichloe
¨ es ucae endophy e-in ec ed ille s o na u ally (E+) and
manipula i ely (ME+) endophy e-in ec ed Fes uca ub a plan s in F
1
gene a ion. The means a e calcula ed o each mo he plan (n = 10).
E o ba s show S.E.
doi:10.1371/jou nal.pone.0011395.g002
Figu e 3. Loss o in ec ion in sexually p oduced seedlings. P opo ional (%) loss o endophy e in ec ion in Fes uca ub a seedlings (F
2
gene a ion). O he 47 endophy e-in ec ed (E+) mo he plan s (F
1
gene a ion), 14 p oduced o sp ing o exclusi ely endophy e-in ec ed seedlings
( o al = 20), 13 p oduced o sp ing o exclusi ely endophy e- ee seedlings ( o al = 19), while 20 mo he plan s p oduced p ogenies consis ing o bo h
E+and E- seedlings (44 and 37, espec i ely) in he F
2
gene a ion. Simila ly, h ee and ou ou o 12 manipula i ely endophy e-in ec ed (ME+) mo he
plan s p oduced o sp ing o exclusi ely endophy e-in ec ed ( o al = 3) o – ee ( o al = 4) seedlings, and i e o he mo he plan s p oduced
p ogenies consis ing o bo h E+and E- seedlings (10 and 13, espec i ely) in he F
2
gene a ion. In o al, E+mo he plan s p oduced 63 and 57, and
ME+mo he plan s, 21 and 16 es ablished E+and E- seedlings, espec i ely.
doi:10.1371/jou nal.pone.0011395.g003
Gene ic Misma ch & Endophy es
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s a us o he plan s was e i ied in all di e en phases o he s udy
by g owing he ungi ou om su ace s e ilized lea es, and by
using a issue p in immunoblo (TPIB) assay [35].
Common ga den expe imen s
These expe imen s we e designed o examine (1) he success o
e ical ansmission, and (2) he ela i e impo ance o endophy e
in ec ion and ma e nal e ec s on hos g ass pe o mance, which
can be seen as po en ially adap i e esponses o he ma e nal
en i onmen s [36]. He e, na u ally and a i icially in ec ed and
endophy e- ee seedlings, i.e. E+,ME+, E- and ME-, espec i ely
we e ansplan ed om he g eenhouse o a common ga den a
Ruissalo Bo anical Ga den, Tu ku, in 2003. In o al, 679 seedlings
comp ising 134 and 72 E+, 39 and 35 ME+, 184 and 139 E- and
35 and 41 ME- o 75 ma e nal plan s o igina ing om meadows
(48 plan s) o i e banks (27 plan s), espec i ely, we e andomly
assigned wi hin 19 blocks and plan ed in sand.
Success o e ical ansmission. To de e mine he success
o e ical ansmission in ege a i e ille s (F
1
gene a ion), ou ille s
we e gen ly de ached om 10 E+plan s (ma e nal en i onmen : 6
and 4 om meadows and i e banks, espec i ely) and om 10
ME+plan s (ma e nal en i onmen : 4 and 6 om meadows and
i e banks, espec i ely) g owing in he common ga den in 2007.
The ille s we e hen ansplan ed sepa a ely in o 7.567.5 cm po s
con aining sand and g own in he g eenhouse o wo mon hs, a e
which hei in ec ion s a us was de e mined om all o a leas 10 o
he ille s, depending on hei size.
To de e mine any ansgene a ional educ ion in in ec ion in
he sexually p oduced F
2
gene a ion, we collec ed all seeds o
expe imen al plan s in Augus 2004, ge mina ed and plan ed
hem, and de e mined he in ec ion s a us o he es ablished
seedlings.
Impo ance o endophy e symbiosis and ma e nal
e ec s. To examine ela i e impo ance o endophy e
symbiosis and ma e nal e ec s, plan pe o mance was examined
as biomass in 2004, including he numbe o in lo escences
p oduced each yea (2004–2008).
S a is ical analyses. We used Chi-squa e analyses (x
2
– es )
o es he e ec s o endophy e manipula ion (na u al o
in oduced) on loss o in ec ion du ing ege a i e g ow h and
seed p oduc ion, as well as any di e ences in endophy e
inocula ion success among o iginally E- seed amilies and
endophy e isola es. In o al, 23 o iginally E- seed amilies and 33
endophy e isola es p oducing a leas i e es ablished seedlings
we e included in o he s a is ical analyses o mee he equi emen s
o he x
2
- es [37]. The e ec s o he o iginal habi a (meadow o
i e bank) and endophy e s a us on one yea biomass p oduc ion
in he common ga den was analysed wi h he GENMOD
p ocedu e, using gamma dis ibu ion and powe (21) as a link
unc ion. We examined in lo escence p oduc ion in common
ga den g asses in 2004–2008 using wo me hods: i s ly, wi h a
epea ed measu es model and hen sepa a ely o each yea wi h
GENMOD (nega i e binomial dis ibu ion and log-link unc ion).
The a iables o iginal habi a , endophy e s a us and hei
in e ac ion we e used as ixed ac o s in he s a is ical models.
Analyses we e done wi h he SAS so wa e package e sion 9.1
(SAS Ins i u e, Ca y, NC, USA).
Resul s
Ve ical ansmission o he ungus is impe ec in na u e. O he
110 plan s collec ed in he ield, 58 hos ed he endophy ic ungus,
o which 23 p oduced bo h endophy e-in ec ed and endophy e-
ee seedlings in he F
1
p ogeny. This demons a es ha he ungal
in ec ion was los in some seedlings o he o sp ing in 40% o he
endophy e-in ec ed ma e nal amilies in na u e.
Endophy e manipula ion ials sugges ha he gene ic compa -
ibili y be ween he ungus and he hos d i es he symbiosis. One
i h o he F
1
p ogenies did no accep he endophy e a all,
inocula ion success a ying om 0 o 88% among he hos plan
p ogenies (x
2
= 42.25, d = 22, p = 0.008) (Fig. 1 a). Simila ly,
inocula ion a emp s we e unsuccess ul wi h nine ungal isola es,
and inocula ion success among he 49 ungal isola es a ied om
0–100% (Fig. 1 b). Howe e , he di e ence in in ec i i y among
endophy e isola es emained s a is ically insigni ican (x
2
= 35.40,
d = 32, p = 0.311) when only he cases o a leas i e es ablished
seedlings we e included in he analyses o mee he equi emen s o
he x
2
- es [37]. Ne e heless, he inocula ion success a ied om
0–60% in hese 33 cases (Fig. 1 b). These esul s sugges ha a
ealized asso men o he endophy e-g ass symbioses is p ima ily
de e mined by he hos g ass geno ype and can be pa ly con ined
o well-ma ched geno ype-geno ype combina ions o he endo-
phy es and he hos g asses.
In he common ga den and g eenhouse expe imen s, he loss o
in ec ion was p onounced and appea s o be pa ly a ibu able o
he gene ic misma ch be ween he ungus and he hos . Fi s ly, he
in ec ion was los in 33% o ege a i e g ass ille s (Fig. 2) and 46%
o sexually p oduced seedlings (Fig. 3), which demons a es ha
he loss o in ec ion can be subs an ially mo e equen han has
been commonly assumed in p e ious li e a u e [4,6,16,24,27] and
highe in seeds p oduced by ou c ossing compa ed o clonally
p oduced plan s. Secondly, consis en wi h he hypo hesis o
gene ic misma ch, ungal g ow h in ege a i e ille s was 27%
lowe in ME+ma e nal amilies compa ed o E+ amilies (Fig. 2;
x
2
= 12.27, d =1, P,0.0005). Meanwhile sexually p oduced
Figu e 4. In lo escence p oduc ion o plan s. The numbe o
in lo escences o in ec ed (E+), manipula i ely in ec ed (ME+), endo-
phy e- ee (E-) and manipula i ely endophy e- ee (ME-) Fes uca ub a
plan s (F
1
gene a ion) o igina ing om meadow and i e bank habi a s
(pa en al gene a ion) g owing in a common ga den be ween 2004–
2008 (yea ly means 6S.E.).
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Gene ic Misma ch & Endophy es
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o sp ing o E+and ME+plan s comp ised o only E-, a mix u e o
bo h E+and E- and only E+p ogenies in simila p opo ions
(Fig. 3; x
2
= 0.1835, d =2, P,0.9123). The g ea e in ec ion
ins abili y in ege a i e ille s in ME+plan s can be expec ed
because he no el ungal-g ass geno ype combina ions we e no
es ed and selec ed o by na u al selec ion. Ins ead, acco ding o
he hypo hesis, ansgene a ional ins abili y should be equal in
ME+and E+plan s in common ga den whe e c oss-pollina ion
eely occu s be ween he expe imen al plan s and nea by wild
g asses, he eby inc easing gene ic misma ch be ween he ungus
and he hos . This c ea es an un i endophy e-g ass geno ype
combina ion and des abilizes he symbiosis.
In 2004, he biomass o common ga den g asses was a ec ed by
habi a (x
2
=12.23,d =1,P,0.0005), bu no by endophy e in ec ion
(x
2
= 1.61, d =3, P,0.6571) o hei in e ac ion (x
2
=0.74, d =3,
P,0.8646). Meadow g asses showed a sligh ly highe biomass
(mean6SE: 0.46560.012 g) han i e bank g asses (mean6SE:
0.40260.013 g). The emo al o he abo e-g ound biomass o plan s
dec eased numbe o in lo escences in he ollowing yea 2005 (Fig. 4).
The e was a h ee-way in e ac ion be ween yea , habi a
(meadow o i e bank) and endophy e s a us in in lo escence
p oduc ion o common ga den ed escues (Table 1). In each s udy
yea , meadow g asses p oduced mo e in lo escences han hose
om he i e bank (Table 2, Fig. 4). Endophy e-in ec ed g asses
om bo h habi a s p oduced mo e in lo escences in 2005 compa ed
o unin ec ed plan s. In 2008, endophy e-in ec ed g asses om
meadows again p oduced mo e in lo escences han unin ec ed
g asses. By compa ison, endophy e- emo ed (ME-) g asses p o-
duced a ewe in lo escences compa ed o o he s (Table 2, Fig. 4).
Discussion
Ou esul s demons a e ha gene ic misma ches, ma e nal
e ec s and loss o in ec ions occu commonly in endophy e-g ass
in e ac ions and may pa ly explain hose di e ences de ec ed in
in ec ion equencies and gene ic s uc u es among na u al g ass
popula ions [6,36]. Fo example, seeds p oduced by ou c ossing
should ha e a high equency o misma ches be ween he ungus
and he g ass and hus pionee g ass popula ions ha ing a la ge
po ion o newly es ablished indi iduals should ha e lowe
in ec ion equencies han olde popula ions. The highe equen-
cies o endophy e-in ec ed g asses de ec ed in meadows compa ed
o sandy i e banks suppo his iew [27]. Meadows a e mo e
s able and e ile en i onmen s, whose g ass popula ions a e olde
and well es ablished by clonal sp ead. By con as , i e bank
popula ions su e almos annual dis u bance due o sp ing
looding [27,38]. Fu he mo e, ou esul s indica e ha he
ma e nal en i onmen may s ongly a ec in lo escence p oduc-
ion o he g asses wi h a ime lag and he eby he dispe sal and
compe i i e abili y o endophy e-in ec ed plan s. As p edic ed by
he geog aphic mosaic heo y o co-e olu ion [11], endophy e–
plan in e ac ions appea o p ojec o he ho - and cold-spo s o
selec i e p essu es in an ecosys em. Acco dingly, endophy es may
p o ide selec i e ad an ages o he hos in some en i onmen s
[27,39], which a e occupied by he locally mos i ungus–plan
geno ype combina ions wi hin a popula ion. Consequen ly, in
na u e he es ablished endophy e-hos combina ions clea ly
ep esen only a ac ion o he a ailable gene ic a ia ion in he
popula ions.
The agili y o endophy e in ec ions de ec ed in his wo k
ques ions he s ic mu ualis ic na u e o endophy e-g ass symbiosis
and sugges s ha he high equencies o endophy e-in ec ed plan s
in suba c ic ed escue popula ions may only pe sis i he selec i e
ad an age o endophy e in ec ion o he hos plan is high. Because
he endophy e may inc ease i s dis ibu ion and i ness p ima ily by
inc easing he plan ’s alloca ion o emale unc ions, we coun ed he
numbe o in lo escences in he common ga den expe imen o e a
i e yea pe iod. On a e age 44% o plan s p oduced in lo escences
each yea . Endophy e in ec ion inc eased in lo escence p oduc ion
o g asses in some yea s ega dless o he manipula ion s a us o he
in ec ion. This di e ence in in lo escence p oduc ion be ween
endophy e-in ec ed and endophy e- ee g asses was mo e p o-
nounced and equen in meadow g asses (Fig. 4). This may pa ially
explain he highe in ec ion equencies de ec ed in meadows
compa ed o i e banks [27]. In e es ingly, he p oduc ion o
in lo escences in ME- plan s declined owa ds he end o he
expe imen , sugges ing ha he loss o he endophy e a e a long co-
e olu iona y ela ionship may be disad an ageous o hos plan
i ness when long- e m ep oduc i e success is aken in o accoun .
These esul s emphasize he impo ance o bo h long- e m
expe imen s and demog aphic eco ds om na u al popula ions
in ecological and e olu iona y s udies.
Ou indings ha e heo e ical and me hodological implica ions,
wi h po en ial economic alue in ag onomy as well. Fi s ly, hese
Table 1. Repea ed measu es analysis o in lo escence
p oduc ion o Fes uca ub a g asses in a common ga den.
Explana o y a iable d x
2
p
Habi a (H) 1 26.00 ,.0001
Endophy e s a us (E) 3 12.22 0.0067
H*E 3 0.98 0.8052
Time (T) 4 71.06 ,.0001
E*T 12 22.25 0.0348
H*T 4 11.93 0.0179
H*E*T 12 25.22 0.0138
doi:10.1371/jou nal.pone.0011395. 001
Table 2. E ec s o o iginal habi a and endophy e s a us on in lo escence p oduc ion o Fes uca ub a g asses g own in a common
ga den in di e en yea s.
2004 2005 2006 2007 2008
Exp. Va . d x
2
px
2
px
2
px
2
px
2
p
Habi a (H) 1 24.1 ,0.001 3.3 0.068 10.6 0.001 21.2 ,0.001 21.4 ,0.001
Endo (E) 3 1.7 0.641 15.4 0.002 5.5 0.138 2.9 0.416 10.8 0.013
H*E 3 2.9 0.401 5.3 0.153 0.7 0.880 1.3 0.719 12.5 0.006
Abb e ia ions: Exp.Va . = explana o y a iable, Endo = Endophy e s a us.
doi:10.1371/jou nal.pone.0011395. 002
Gene ic Misma ch & Endophy es
PLoS ONE | www.plosone.o g 5 June 2010 | Volume 5 | Issue 6 | e11395

esul s illus a e he impo ance o gene ic incompa ibili y in ou -
c ossing g ass popula ions and ma e nal e ec s as des abilizing
o ces in endophy e-g ass symbioses. These o ces may ac as
bu e ing mechanisms agains compe i i e endophy e-g ass geno-
ype combina ions, po en ially domina ing he popula ions and
g assland communi ies. Secondly, gene ically de e mined esis-
ance o endophy ic ungi and gene ic misma ch ha cons ains
combina ions o ungi and hos g ass, should be acknowledged in
b eeding p og ammes aimed a imp o ing ag icul u ally aluable
cha ac e is ics o cul i a s such as highe yield and esis ance o
he bi o es and pa hogens.
Acknowledgmen s
We hank Ke in O’B ien, Alan Gange, Dennis Hansen and one
anonymous e iewe o aluable commen s on he manusc ip .
Au ho Con ibu ions
Concei ed and designed he expe imen s: KS PRW MH. Pe o med he
expe imen s: KS PRW MH. Analyzed he da a: KS PRW MH. W o e he
pape : KS PRW MH.
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Gene ic Misma ch & Endophy es
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