En i onmen al condi ions and hos plan o igin o e ide
endophy e e ec s on in e eb a e communi ies
Salla-Riikka Ves e lund &Ma jo Helande &
S anley H. Fae h &Te ho Hy önen &Ka i Saikkonen
Recei ed: 15 Decembe 2010 /Accep ed: 5 Janua y 2011 /Published online: 29 Janua y 2011
#The Au ho (s) 2011. This a icle is published wi h open access a Sp inge link.com
Abs ac Sys emic ungal endophy es o g asses can p oduce
high concen a ions o alkaloids ha a e known o de e
in e eb a e he bi o es and educe hei abundance, especially
in ag onomic g asses. G ass endophy es may also in luence
in e eb a e communi y di e si y and composi ion. He e, we
examined in a common ga den expe imen wi h wild all
escue plan s and he ag onomic cul i a Ken ucky 31, whe he
in ec ion by Neo yphodium coenophialum, he gene ic back-
g ound (o igin) o he hos plan , abio ic ac o s, and hei
in e ac ions a ec ed he in e eb a e communi ies li ing on
all escue. We collec ed a o al o 18650 in e eb a es om
he 480 all escue plan s, iden i ied hem o 97 mo phological
axa (mainly o amily le el) ep esen ing i e eeding guilds
(he bi o es, de i i o es, omni o es, p eda o s and pa asi-
oids). In con as o pas li e a u e, endophy e in ec ion did
no a ec abundances o any axon o eeding guild, o
axonomic di e si y and he composi ion o he in e eb a e
communi y. Ins ead, he in e eb a e communi y o na i e all
escue appea s o be p ima ily d i en by en i onmen al
condi ions and niche di e en ia ion among axonomical
g oups o in e eb a es. We p opose ha communi y
app oaches a e equi ed o unde s and o he ole o
endophy es on a h opod abundances and di e si y in na u e.
Keywo ds Endophy ic ungi .Tall escue .In e eb a e
di e si y and communi y s uc u e .Func ional guild .
He bi o y.Gene ic a ia ion
In oduc ion
Asexual Neo yphodium endophy es ( amily Cla icipi aceae)
o m symbio ic ela ionships wi h many cool-season g asses
belonging o he sub- amily Pooidae (Clay 1988,1990).
In ec ions a e sys emic and he endophy e is ansmi ed
e ically o he nex gene a ion h ough seeds (Scha dl e al.
2004;ClayandScha dl2002). Tall escue (Schedono us
phoenix (Scop. Holub.) [ = Lolium a undinaceum (Sch eb.)
Da bysh. = Schedono us a undinaceus (Sch eb.) Dumo ])
has been widely used as o age and u g ass in he
Uni ed S a es o decades (Ball e al. 1993). Thus, one o
he mos s udied g ass–endophy e associa ions is he N.
coenophialum and all escue symbiosis (Saikkonen e al.
2006,2010). Tall escue cul i a s a e domina ed by a
widely-adap ed cul i a named “Ken ucky 31”(he ea e
e e ed o as K-31), which has a long g owing season and
is esis an o pes s, d ough , poo soil condi ions, and
a ia ions in soil pH (Ball e al. 1993). Based on he
esea ch o his g ass–endophy e sys em, he ela ionship
be ween he endophy ic ungus and i s hos has gene ally
been hough o be mu ualis ic (Clay 1988; Clay e al.
1993; Saikkonen e al. 2006; Scha dl and Phillips 1997).
Recen s udies ha e shown, howe e , ha his ela ionship
can a y om mu ualism o an agonism, depending on he
geno ype o he ungus and he hos as well as en i on-
men al condi ions, especially in na i e g asses (Cheplick
e al. 1989; Cheplick and Fae h 2009; Fae h 2002;Fae h
and Saikkonen 2007; Fae h and Sulli an 2003). Saikkonen
e al. (1998,2004,2006) he e o e p oposed ha he
S.-R. Ves e lund (*):M. Helande
Sec ion o Ecology, Depa men o Biology, Uni e si y o Tu ku,
Tu ku FI-20014, Finland
e-mail: [email p o ec ed]
S. H. Fae h
Depa men o Biology,
Uni e si y o No h Ca olina a G eensbo o,
G eensbo o, NC 27402, USA
T. Hy önen :K. Saikkonen
MTT Ag i ood Resea ch Finland, Plan P o ec ion,
Jokioinen FI-31600 Finland
Fungal Di e si y (2011) 47:109–118
DOI 10.1007/s13225-011-0089-x
p e ailing concep o endophy es as mu ualis s is likely
his o ical and sys em based a he han based on e idence
om na u al popula ions.
In he case o he all escue–N. coenophialum symbiosis,
much o he esea ch has been done in he Uni ed S a es on
ag onomic cul i a s such as K-31 (Saikkonen e al. 2006),
al hough he o igins o his g ass a e in Eu asia. In hese
ag onomic cul i a s plan ed ou side hei na i e dis ibu ional
ange, Neo yphodium is widely known o cause de imen al
e ec s (e.g., oxicosis) on e eb a e g aze s in high-nu ien
ag onomic en i onmen s (Ball e al. 1993; Clay 1989,1990;
Saikkonen e al. 2006,2010; Scha dl and Phillips 1997).
These e ec s a e ela ed o high concen a ions o alkaloids
(Clay 1990; Lyons e al. 1986), which a e known o de e
bo h e eb a e and in e eb a e he bi o es (Bacon 1995;
Bacon e al. 1977; Bazely e al. 1997; Siegel and Bush 1996,
1997; Vica i e al. 2002). Because alkaloids a e nu ien - ich
compounds, hei syn hesis has cos o o he basic plan
g ow h and ep oduc i e unc ions (Fae h 2002; Fae h and
Bul man 2002; Fae h and Fagan 2002). These cos s may
ou weigh he bene i s o he endophy e in ec ion in mos
en i onmen s, bu pa icula ly so in nu ien -poo en i-
onmen s in na u e (Ahlholm e al. 2002; Fae h 2002;
Leh onen e al. 2005). Thus, in i s na i e habi a , in ec ed
wild all escue may p oduce lowe le els and ewe ypes
o alkaloids han i s cul i a ed and selec i e-b ed a ie ies
in nu ien - ich en i onmen s in he in oduced ange
(Saikkonen e al. 1998,2010;SiegelandBush1996;bu
see Piano e al. 2005).
Recen e idence suppo s his idea: (1) he le els and
composi ion o alkaloids p oduced a ies among ungal
species and geno ypes (e.g., Piano e al. 2005; Siegel and
Bush 1997), also (2), alkaloid ypes and le els in na u al
popula ions a y mo e han hose in ag onomic g asses
(Bony e al. 2001; Fae h and Saikkonen 2007), (3) he
numbe o non- oxic endophy e-in ec ed g asses exceed
oxic ones (Fae h 2002), and (4) in some cases, in ec ion
dec eased, a he han inc eased, he he bi o e esis ance o
he hos plan (Fae h and Shocha 2010; Jani e al. 2010;
Saikkonen e al. 1998; Schul hess and Fae h 1998).
Al ough well-s udied in ag onomic cul i a s such as
K-31 in in oduced a eas, he in e ac ions be ween all
escue and Neo yphodium endophy es a e s ill la gely
igno ed in hei na i e ange in Eu ope (Saa i e al. 2010;
Zabalgogeazcoa and Bony 2005), p obably because all
escue is no a p e e ed li es ock o age g ass (Niemeläinen
e al. 2001) and li es ock oxicosis is a e (Zabalgogeazcoa
and Bony 2005). The na u e and ecological impo ance o
he all escue–N. coenophialum symbiosis may be di e en
in i s na i e ange (Saikkonen 2000; Saikkonen e al. 1998;
Siegel and Bush 1996).
We examined whe he he N. coenophialum endophy e
in ec ion and he o igin o he hos plan as well as abio ic
ac o s and hei possible in e ac ions a ec he in e e-
b a e communi y li ing on all escue. Besides he bi o es,
ungal endophy es may also a ec de i i o es (e.g.,
Lemons e al. 2005) and he na u al enemies o he bi o es
(Fae h and Shocha 2010; Ha ley and Gange 2009;Janie
al. 2010; Omacini e al. 2001) o ende he bi o es mo e
o less suscep ible o na u al enemies by a ec ing hei
a ack a es (Ben ey and Denno 1997; Saa i e al. 2010)
and delaying he bi o e de elopmen (e.g. B een 1994;
Clay e al. 1985; Popay and Rowan 1994). Howe e , he e
a e only a ew s udies ha ha e conside ed he impac o
g ass endophy es on a h opod communi ies o unc ional
g oups (e.g., A khami and Rudge s 2009; Fae h and
Shocha 2010;Janie al.2010). In his s udy, we used a
whole-in e eb a e communi y su ey o a con olled
common ga den expe imen o es how in e eb a e
di e si y and communi y s uc u e, and he numbe o
indi iduals in unc ional in e eb a e axa and guilds
di e s be ween (i) endophy e in ec ed (E+), endophy e
ee (E-), and manipula i ely endophy e- ee (ME-) all
escue, (ii) hos plan s o di e en o igin (wild popula ions
om Åland, Go land, coas al Sweden and one ag onomical
cul i a , K-31 om USA), and (iii) hos plan s g owing
in di e en abio ic en i onmen s (nu ien and wa e
ea men s). Based on he pas s udies on de ensi e
endophy e-g ass mu ualism (Saikkonen e al. 2010)and
he ew ecen s udies on how endophy es s uc u e
a h opod communi ies in all escue (Rudge s and Clay
2008), we p edic ed ha : (1) endophy e in ec ion dec eases
in e eb a e di e si y and communi y s uc u e, (2) he
abundances o plan eeding in e eb a es (he bi o es,
omni o es and de i i o es) a e highly a iable, bu in
gene al, lowe in endophy e in ec ed (E+) hos plan s
compa ed wi h endophy e- ee plan s, pa icula ly in he
e ilized plan s, (3) he ag icul u al cul i a (K-31) shows
s ong de e ence o plan eeding in e eb a es, and (4)
endophy e in ec ion may bo h posi i ely and nega i ely
a ec he na u al enemies o he bi o es (p eda o s and
pa asi oids).
Ma e ials and me hods
Plan and seed ma e ial
To es he e ec o in ec ion, hos plan o igin, and
en i onmen al ac o s (wa e and nu ien ea men s), in
Augus 2005, we collec ed seeds om mul iple na u al
all escue popula ions by he Bal ic Sea in locali ies
ha we e geog aphically sepa a ed om each o he by
app oxima ely 500 km. These we e he island o Åland
(8 popula ions), he island o Go land (9 popula ions),
and he wes coas o Sweden (6 popula ions). 10 o 50
110 Fungal Di e si y (2011) 47:109–118
indi iduals we e collec ed om each popula ion, and
h ee seeds om each plan indi idual we e s ained o
mic oscopic examina ion o he endophy e in ec ion
s a us (Saha e al. 1988). Neo yphodium coenophialum
in ec i i y a ied be ween 85–100% in all all escue
popula ions om he h ee loca ions. Unin ec ed (E-) and
in ec ed (E+) seeds we e combined sepa a ely om
popula ions wi hin each o he h ee s udy a eas (Åland,
Go land, and coas al Sweden). In o he wo ds, we pooled
all E- seeds and hen all E+ om he popula ions wi hin
each loca ion o c ea e h ee ba ches o E- seeds and h ee
ba ches o E+ seeds ha ep esen ed he h ee geog aphic
o igins. In addi ion o plan s om na u al all escue
popula ions, we used E+ and E- K-31 ( om T. Phillips,
Uni e si y o Ken ucky) cul i a seeds in ou expe i-
men . To es he ole o he endophy e on in e eb a e
communi ies while con olling o plan geno ypic
backg ound, we expe imen ally emo ed he endophy e
om po ion o E+ seeds (manipula i ely endophy e-
ee plan s = ME-). To kill he ungus while he seeds
emained iable, he E+ seeds we e hea – ea ed by
keeping he seeds in wa m wa e (56-57°C) o 10–
20 min.
All all escue seeds om na u al popula ions, K-31
cul i a and endophy e- emo ed seeds we e ge mina ed on
mois issue pape in Pe i-dishes in a g eenhouse and
plan ed 7 days a e ge mina ion o indi idual po s wi h
sand and pea mix u e.
Table 2 The e ec s o endophy e s a us (E+ = endophy e in ec ed, E- = endophy e- ee, and manipula i ely endophy e ee = ME-), wa e and nu ien ea men s (C = con ol, N = nu ien , W =
wa e , and WN = wa e + nu ien ), plan o igin (A = Åland, G = Go land, and S = coas al Sweden; K = cul i a “Ken ucky 31”) and plan biomass on he abundances o dip e ans, mi es (Aca i),
Hymenop e ans, collembolas and Coleop e ans
Taxon Feeding guild Endophy e s a us (E) T ea men (TRT) Plan o igin (PO) E*TRT E*PO TRT*PO Plan biomass
d =2 d =3 d =3 d =6 d =6 d =9 d =1
F p F p F p Fp Fp Fp F p
Dip e a he bi o ous 0.20 0.8202 2.34 0.0727 2.15 0.0931 2.30 0.0337 0.59 0.7402 2.57 0.0070 9.21 0.0026
de i i o ous 0.84 0.4317 11.62 <0.0001 3.04 0.0291 0.92 0.4807 1.06 0.3846 2.36 0.0133 5.47 0.0199
omni o ous 1.04 0.3540 0.97 0.4091 1.29 0.2791 3.04 0.0063 0.90 0.4948 1.18 0.3058 1.59 0.2077
pa asi ic 0.06 0.9398 0.97 0.4072 1.63 0.1820 1.40 0.2122 0.99 0.4289 0.77 0.6458 5.75 0.0169
p eda o y 1.52 0.2190 2.57 0.0537 1.07 0.3628 1.30 0.2541 0.45 0.8420 0.68 0.7289 0.31 0.5761
Aca i omni o ous & pa asi ic 1.16 0.3141 3.76 0.0110 0.07 0.9743 0.41 0.8735 1.69 0.1220 0.61 0.7885 4.66 0.0315
Hymenop e a pa asi ic 2.13 0.1204 0.68 0.5659 4.76 0.0028 0.51 0.7970 0.73 0.6279 1.48 0.1518 0.59 0.4446
A aneae p eda o y 0.47 0.6260 1.95 0.1213 1.16 0.3255 0.64 0.6975 1.05 0.3911 0.93 0.5025 4.13 0.0429
Collembola de i i o ous 0.97 0.3785 11.91 <0.0001 3.14 0.0253 2.68 0.0146 0.29 0.9404 0.75 0.6660 10.39 0.0014
Coleop e a de i i o ous 0.16 0.8514 23.63 <0.0001 3.10 0.0268 1.95 0.0716 0.31 0.9322 2.51 0.0084 0.07 0.7964
p eda o y 2.67 0.0708 18.81 <0.0001 1.28 0.2792 0.68 0.6669 1.60 0.1455 1.77 0.0730 2.85 0.0923
Table 1 In e eb a e axa collec ed om he expe imen al plan s
Taxon Numbe o
indi iduals
Feeding guild
Dip e a 1393 he bi o ous
704 de i i o ous
328 omni o ous
25 p eda o y
3 pa asi ic
Hymenop e a 46 he bi o ous
606 pa asi ic
Collembola 8360 de i i o ous
Hemip e a 197 he bi o ous
51 p eda o y
Homop e a 37 he bi o ous
Coleop e a 28 he bi o ous
379 p eda o y
589 de i i o ous
A aneae
(A achnida)
281 p eda o y
Aca i (A achnida) 4017 omni o ous / pa asi ic
Thysanop e a 62 (guild no iden i ied)
Fungal Di e si y (2011) 47:109–118 111
Field expe imen
To es he ole o endophy e in ec ion, plan geog aphic
o igin and en i onmen al ac o s, a common ga den ield
expe imen was es ablished a Bo anical Ga den, Uni e -
si y o Tu ku, Finland in 2004. The s udy si e is a he
edge o he no he n dis ibu ion ange o na u al all
escue popula ions and has been in cul i a ion in he
pas . I was illed in he summe 2004 wi hou nu ien
applica ion. The expe imen al a ea was enced o p e en
la ge e eb a es (e.g., abbi s, dee ) om b owsing he
plan s. Howe e , smalle e eb a es (e.g., oles) and
in e eb a es we e allowed o eely access he a ea. The
space be ween expe imen al plan s was ei he mowed,
hand weeded o sp ayed wi h he bicide wo imes du ing
he g owing season o p e en in e speci ic compe i ion
in he ield.
The expe imen al design was a andomized block
consis ing o 10 blocks, each di ided o 4 plo s. The ou
plo s in each block we e andomly designa ed o one o
he ou ea men s: (i) con ol (C) ecei ing only
ambien wa e and nu ien s, (ii) wa e ea men (W)
wi h 3 li es o wa e applied o each plan sepa a ely
h ee imes a week om June o Augus , (iii) nu ien
ea men (N) whe e 1dl o N-P-K- e ilize (Nu men Y2,
Kemi a KnowHow,[N-P-K/20-6-6])/plan was applied
wo imes du ing he g owing season, and (i ) wa e –
nu ien ea men (WN) combining bo h wa e and
nu ien applica ions. The ea men s we e applied du ing
he pe iod o 2005–2006.
Tall escue plan s wi h 2-3 ille s we e plan ed in Augus
2004 abou 0.5 me e s apa om each o he and om he
edge o he plo . Fo y plan s om each o igin (na u al
popula ions A = Åland, G = Go land, and S = coas al
Sweden; cul i a s K = “Ken ucky 31”) and in ec ion s a us
(E+, E-, ME-) we e andomly chosen. Thus, he e we e 12
plan s in each o he 40 plo s o a o al o 480 plan s used
in he p esen s udy. The in ec ion s a us o all indi idual
plan s was con i med in 2006 ia seed s aining (Saha e al.
1988). The biomass o he abo e-g ound plan pa s was
emo ed, d ied and weighed in au umn a he end o he
g owing season 2006.
Collec ion and iden i ica ion o in e eb a es
In e eb a es we e collec ed om each plan indi idual
wi h an Insec Vo is Vaccuum® sample (Bu ka d L d.,
UK) in July 2006. E e y plan was acuumed in he
same way o 10 s om he middle o he plan . The
samples we e placed in o eclosable plas ic bags and
ozen immedia ely a e sampling. In e eb a es we e
hen la e coun ed, iden i ied o amily le el unde a
mic oscope, and assigned o he ollowing i e eeding
guilds based on he key amily and species cha ac e is ics in
li e a u e: he bi o es, omni o es, de i i o es, p eda o s and
pa asi oids (Table 1).
S a is ical analyses
We used ANCOVA (wi h plan biomass as a co a ia e)
in he Mixed model p ocedu e o SAS s a is ical
so wa e (SAS U ili ies 9.1) o analyze he e ec s o
endophy e s a us (E+, E-, and ME-), wa e and nu ien
ea men s (W, N, WN, and C), plan o igin (A, G, K,
S), and block (1–10) on he abundances o in e eb a es
in he eeding guilds (he bi o es, omni o es, de i i-
o es, p eda o s and pa asi oids) and axonomical
g oups wi h su icien sample sizes o meaning ul
s a is ical analyses (he bi o ous, de i i o ous, omni o-
ous, pa asi ic, and p eda o y dip e ans; Aca i; Hyme-
nop e ans; spide s; collembolas, and de i i o ous and
p eda o y Coleop e ans) T ea men , o igin, endophy e,
and hei possible in e ac ions we e conside ed o be
ixed ac o s in all models, whe eas block was a
andom ac o . Plan biomass was used as a co a ia e,
because plan size may in luence in e eb a e abundan-
Table 3 The e ec s o endophy e s a us (E+ = endophy e in ec ed,
E- = endophy e- ee, and manipula i ely endophy e- ee = ME-),
wa e and nu ien ea men s (C = con ol, N = nu ien , W = wa e , and
WN = wa e + nu ien ), plan o igin (A = Åland, G = Go land, and
S = coas al Sweden; K = cul i a “Ken ucky 31”) and plan biomass on
abundances o he bi o es, de i i o es and p eda o s
He bi o es De i i o es Omni o es Pa asi oids P eda o s
d Fp F p Fp Fp F p
Endophy e s a us (E) 2 0.35 0.7036 0.80 0.4484 0.29 0.8330 2.14 0.1192 2.31 0.1007
T ea men (TRT) 3 3.10 0.0268 15.05 <0.0001 0.71 0.5471 0.63 0.5987 15.38 <0.0001
Plan o igin (PO) 3 1.61 0.1870 3.99 0.0080 0.52 0.5932 4.59 0.0036 1.04 0.3730
E * TRT 6 2.62 0.0169 2.63 0.0165 0.50 0.8089 0.55 0.7674 0.68 0.6681
E * PO 6 0.74 0.6199 0.26 0.9565 0.87 0.5156 0.75 0.6119 1.04 0.3987
TRT * PO 9 1.94 0.0449 0.72 0.6885 0.44 0.9142 1.46 0.1591 1.45 0.1662
Plan biomass 1 9.67 0.0020 10.28 0.0015 0.04 0.8338 0.78 0.3781 3.22 0.0734
112 Fungal Di e si y (2011) 47:109–118
ces. Plan size was signi ican ly inc eased by wa e ing
and e iliza ion (d =3, F=17.07, p<0.0001)(C: mean=
395g,SE=16.4;N:mean=414g,SE=22.1;W:mean=
422 g, SE=15.2; WN: mean=587 g, SE=24.2) excep in
he case o he K- 31 cul i a . Resul s on plan g ow h and
pe o mance will be epo ed and discussed in mo e de ail
elsewhe e.
The e ec s o endophy e s a us (E+, E-, and ME-),
wa e and nu ien ea men s (W, N, WN, and C), plan
o igin (A, G, K, S) and plan biomass on axonomic
in e eb a e di e si y we e examined in wo ways. Fi s ,
we es ed he e ec s o he explana o y ac o s and hei
in e ac ions on species numbe s and he Shannon
di e si y index by a mixed model analysis o co a iance
Table 4 Means and s anda d e o s (SE) o axonomic g oups o
in e eb a es showing s a is ically signi ican (a) in e ac i e e ec s o
wa e and nu ien ea men s (C = con ol, N = nu ien , W = wa e ,
and WN = wa e + nu ien ) and endophy e s a us (E+ = endophy e
in ec ed, E- = endophy e- ee, and manipula i ely endophy e- ee = ME-),
(b) e ec s o plan o igin (A = Åland, G = Go land, and S = coas al
Sweden; K = cul i a “Ken ucky 31”) and (c) in e ac i e e ec s o wa e
and endophy e s a us (see Table 2)
Taxon
a He bi o ous Dip e a Omni o ous Dip e a Collembola
T ea men Endophy e s a us n mean SE mean SE mean SE
C E+ 39 2.7 2.7 1.2 0.37 9.4 1.76
E- 39 3.4 3.4 0.5 0.14 10.2 2.03
ME- 40 3.7 3.7 0.6 0.12 11.7 2.54
W E+ 39 3.2 3.2 0.7 0.15 20.7 3.27
E- 40 2.6 2.6 0.6 0.13 14.3 2.31
ME- 39 2.1 2.1 0.8 0.25 11.4 1.81
N E+ 32 2.4 2.4 0.6 0.14 21.8 3.36
E- 37 2.4 2.4 0.5 0.13 28.7 5.10
ME- 34 3.6 3.6 0.6 0.13 25.9 3.66
WN E+ 38 3.9 3.9 0.7 0.18 33.7 6.22
E- 34 4.6 4.6 1.6 0.36 18.8 3.87
ME- 34 3.3 3.3 0.5 0.14 22.0 3.80
b De i i o ous Dip e a Hymenop e a Collembola Coleop e a
Plan o igin n mean SE mean SE mean SE mean SE
Åland 118 1.9 0.24 1.8 0.17 24.3 2.34 1.9 0.33
Go land 113 1.65 0.19 1.2 0.17 17.7 2.06 1.2 0.26
K-31 99 1.1 0.14 0.96 0.12 13.9 1.78 0.8 0.14
Sweden 115 1.6 0.17 1.4 0.13 18.4 2.10 1.3 0.18
c He bi o ous Dip e a De i i o ous Dip e a Coleop e a
T ea men Plan o igin n mean SE mean SE mean SE
C Åland 30 2.8 0.47 1.0 0.25 0.3 0.10
Go land 29 3.3 0.60 1.2 0.25 0.3 0.11
K-31 29 3.1 0.44 0.9 0.20 0.4 0.15
Sweden 30 3.6 0.32 1.0 0.26 0.4 0.12
W Åland 28 2.9 0.53 1.8 0.39 0.5 0.15
Go land 30 1.9 0.31 2.0 0.37 0.4 0.09
K-31 30 2.7 0.45 1.0 0.25 0.5 0.16
Sweden 30 3.1 0.64 1.6 0.35 0.7 0.22
N Åland 30 2.9 0.47 1.1 0.22 2.2 0.58
Go land 26 2.8 0.40 1.2 0.31 1.7 0.40
K-31 19 2.6 0.63 1.1 0.27 1.7 0.45
Sweden 28 2.8 0.44 1.3 0.27 1.7 0.33
WN Åland 30 6.1 0.76 3.9 0.72 4.5 1.00
Go land 28 3.6 0.65 2.2 0.52 2.7 0.89
K-31 21 2.2 0.71 1.4 0.38 1.0 0.33
Sweden 27 3.3 0.71 2.6 0.37 2.4 0.53
Fungal Di e si y (2011) 47:109–118 113

(ANCOVA) wi h plan biomass as a co a ia e, using
he Mixed p ocedu e o SAS s a is ical so wa e
(SAS U ili ies 9.1). The plan -speci ic Shannon index
alue (H’) was calcula ed as ollows: H0¼PipilnðpiÞ
whe e p
i
is he p opo ion o indi iduals in he i he
axonomical g oups in he expe imen al plan s. Compa ed
o species numbe o ichness, he ad an age o he
Shannon index is ha i inco po a es he numbe o
axonomical g oups and hei e enness. Second, o
examine he amoun o a ia ion (%) ha endophy e
s a us, wa e and nu ien ea men s and plan o igin
explained in he in e eb a e communi y composi ion, we
used a pa ial Canonical Co espondence Analysis CCA
(Bo ca d e al. 1992) wi h CANOCO 4 so wa e (Te B aak
and Šmilaue 1998). Only he a ia ion explained by
s a is ically signi ican en i onmen al a iables was pa i-
ioned (Økland 1999). The de aul op ions o CANOCO
(excep log x + 1 da a ans o ma ion and downweighing o
a e species) we e used. The signi icance o he i s CCA
axis and he CCA model, as well as each en i onmen al
a iable was e alua ed by Mon e Ca lo pe mu a ion es s
(500 pe mu a ions) in all analyses. Nu ien and wa e
ea men s along wi h plan biomass appea ed o be
signi ican (p<0.01) in CCA.
Resul s and discussion
Recen li e a u e indica es ha ungal endophy es al e
in e eb a e communi ies in bo h ag onomic and wild g ass
popula ions (Rudge s and Clay 2007; Ben ey and Denno
1997; Fae h and Shocha 2010; Ha ley and Gange 2009;
Jani e al. 2010; Lemons e al. 2005; Omacini e al. 2001;
Saa i e al. 2010). Howe e , he Neo yphodium endophy e
in ec ion ailed o in luence he abundances o he axa o
eeding guild, o o e all axonomic di e si y and compo-
si ion o he in e eb a e communi y in ou expe imen al
common ga den s udy o wild all escue plan s collec ed
ac oss he no he n dis ibu ion ange o he species and he
well s udied all escue K-31 cul i a . Ins ead, he in e e-
b a e communi y o na i e all escue in his expe imen
appea s o be p ima ily d i en by en i onmen al condi ions
in e ac ing wi h plan geog aphic o igin.
In e eb a e abundance and communi y composi ion
A o al o 18650 in e eb a es we e collec ed and iden i ied
o amily le el om he expe imen al plan s. Sp ing ails
(Collembola), mi es (Aca i), and lies and midges (Dip e a)
comp ised 48%, 23% and 14% o he indi idual in e eb a es,
espec i ely (a o al o 85%) (Table 1). The es 15% o he
in e eb a es we e Coleop e ans (6%), Hymenop e ans (4%),
spide s (2%), and Hemip e ans (2%). Only one pe cen age o
species emained uniden i ied. 56% and 24% o he
in e eb a e communi y consis ed o de i i o es and pa a-
si oids, espec i ely, because o he high numbe o
de i i o ous Collembola and Aca i mi es and pa asi ic
Hymenop e ans in ou samples (Table 1). Only 10% o all
in e eb a es we e he bi o es, bu his eeding guild was
axonomically he mos di e se comp ising o 42 iden i ied
axa.
E+ plan s did no di e om E- and ME- plan s in he
abundance o any axonomic in e eb a e g oup (Table 2)
o eeding guild (Table 3). Howe e , endophy e in ec ion
a ec ed he abundance o he bi o ous and omni o ous
dip e ans, and collembolas in e ac i ely wi h wa e and
nu ien ea men s. Fo example, he abundance o
he bi o ous dip e ans was highe on wa e ed and e ilized
E- and E+ plan s compa ed o he o he ea men and
in ec ion combina ions, whe eas he abundance o omni -
o ous dip e ans was highes on wa e ed and e ilized E-
plan s, second highes on un ea ed E+ plan s, and lowes
on e ilized E- plan s (Table 4a). In con as o dip e ans,
de i i o ous Collembola (sp ing ails) we e much mo e
ab
CWN
Numbe o he bi o es
0
1
2
3
4
5
6
7
E+
E-
ME-
CWNWN WN
Numbe o de i i o es
0
10
20
30
40
50
E+
E-
ME-
Fig. 1 The e ec s o endophy e
s a us (E+, E-, and ME-) and
wa e and nu ien ea men s
(W, N, WN, and C) on he o al
numbe o he bi o es (a) and
de i i o es (b)
114 Fungal Di e si y (2011) 47:109–118
abundan and appea ed o p e e wa e ed and e ilized E+
plan s (Table 4a; see also Fae h and Shocha 2010).
Likewise, he o al numbe o he bi o es and de i i o es
did no show a common end o p e e ence o a oidance
o E+ o E- plan s in ei he low o high nu ien
en i onmen s (Table 2,Fig.1).
Plan o igin signi ican ly a ec ed he abundances o
de i i o ous Dip e a, Hymenop e a, Collembola and Cole-
op e a (Table 2), as hei mean abundances was highes on
plan s collec ed om Åland and lowes on he cul i a
“Ken ucky 31”in all g oups (Table 4b). In he cases o
Coleop e a and bo h he bi o ous and de i i o ous Dip e a,
abundances a ied among plan o igins in e ac i ely wi h
wa e and nu ien ea men (Table 2), bu we e highes on
plan s om Åland and lowes on he K-31 when he plan
was wa e ed and e ilized (Table 4c). This indica es
di e ences in esis ance among plan geno ypes in di e en
en i onmen s.
Plan size appea s o be posi i ely ela ed o in e eb a e
abundance. Plan biomass explained signi ican ly he numbe s
o he bi o ous, de i o ous and pa asi ic dip e ans, spide s
(A aneae), and mi es (Aca i) (Table 2), and he abundances o
hese axa we e posi i ely co ela ed wi h plan size
excep in he case o pa asi ic dip e ans (he bi o ous Dip e a:
n=445, =0.21, p=<0.0001; de i i o ous: n=445, =0.26,
p=<0.0001; pa asi ic Dip e a: n=445, =0.06, p=<0.2035;
Collembola: n=445, =0.24, p=<0.0001; A aneae: n=445,
=0.13, p=0.0074). Likewise, he o al numbe o bo h
he bi o es and de i i o es posi i ely co ela ed wi h
plan biomass (He bi o es: n=445, =0.22, p=<0.0001;
de i i o es: n=445, =0.26, p=<0.0001).
In e eb a e ichness
In e eb a e ichness ollowed he same ends as axonomic
g oups. Nei he he numbe o axa no Shannon di e si y
index a ied by he in ec ion s a us o he plan (Table 5).
Ins ead, in e eb a e ichness was posi i ely co ela ed wi h
plan size (plan biomass— he numbe o axa: n=444, =
0.27, p=<0.0001; plan biomass— he Shannon di e si y
index: n=444, =0.15, p=0.0016) and i was signi ican ly
highe on e ilized plan s (Table 5,Fig.2).
In e eb a e communi y s uc u e
Canonical Co espondence Analysis (CCA) sugges s ha
in e eb a e communi y well mi o s abio ic en i onmen al
condi ions and he size o he plan . Mos o he a ia ion in
he axonomical composi ion was highly dependen on
nu ien (Axis 1 in Fig. 3a) and wa e (Axis 2 in Fig. 3a)
a ailabili y in he soil. The sum o all canonical eigen alues
was 0.131. The i s axis explained 3.2% o axon a ia ion
and 57.6% o he a ia ion o he axon-en i onmen
ela ionship. In he Mon e Ca lo es , he signi icance o
he i s axis was P=0.002 (F=14.2) and o all axes P=
0.002 (F=2.8). T ea men explained 73.3% o he a ia ion,
ab
CWNWNNW
Numbe o axa
0
2
4
6
8
10
12
CNW
Shannon H'
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
Fig. 2 Mean (±SE) numbe o
axa (a) and he Shannon
di e si y index in wa e and
nu ien ea men s
d Taxon numbe Shannon di e si y index
Fp F p
Endophy e s a us (E) 2 0.88 0.4148 0.37 0.6931
T ea men (TRT) 3 11.05 <0.0001 6.07 0.0005
Plan o igin (PO) 3 1.52 0.2086 0.80 0.4923
E * TRT 6 1.95 0.0714 0.60 0.7268
E * PO 6 1.25 0.2815 1.29 0.2605
TRT * PO 9 1.12 0.3456 1.03 0.4159
Plan biomass 1 12.23 0.0005 4.38 0.0369
Table 5 The e ec s o
endophy e s a us (E+ = endophy e
in ec ed, E- = endophy e- ee, and
manipula i ely endophy e- ee =
ME-), wa e and nu ien ea -
men s (C = con ol, N = nu ien ,
W = wa e , and WN = wa e +
nu ien ), plan o igin (A = Åland,
G=Go land,andS=coas al
Sweden; K = cul i a “Ken ucky
31”) and plan biomass on
iden i ied axon numbe s and he
Shannon di e si y index
Fungal Di e si y (2011) 47:109–118 115
whe eas he p opo ion o he o he ac o s emained
smalle (plan o igin 9.9%, endophy e s a us 7.6%, plan
biomass 6.9%) and s a is ically insigni ican (C: F=7.0, P=
0.002; W: F=5.5, P=0.002; N: F=8.1, P=0.002; NW: F=
3.8, P=0.002; Biomass o he plan : F=1.986, P=0.002; E+:
F=1.161, P=0.2196; E-: F=0.815, P=0.7884; ME-: F=
0.955, P=0.5250; A: F=1.083, P=0.3593; G: F=0.902, P=
0.6727; S: F=0.729, P=0.9022; K: F=0.884, P=0.6966).
Howe e , he e was no common s uc u e in he in e e-
b a e communi y ela ed o endophy e s a us, plan o igin o
wa e and nu ien ea men s ac oss he axonomical g oups o
eeding guilds (Fig. 3). In con as , CCA indica es ha each
axonomical g oup in a eeding guild appea s o p e e
di e en en i onmen s and he axonomical di e si y o a
eeding guild is in ela ion o specializa ion in esou ce
use acco ding o he idea o niche pa i ioning (El on
1927;Hu chinson1961;Richa dse al.2000;Diehl2003).
Fo example, he he bi o e eeding guild was axonomically
mos di e se (42 axa), bu he place o he bi o e axa in he
expe imen al wa e and nu ien en i onmen s we e no
iden ical (Fig. 3b) In o he wo ds, he species clea ly do no
occupy exac ly he same hos ype.
ab
cd
e
Fig. 3 Canonical Co espon-
dence Analysis (CCA) o he
ela ionship be ween axonomical
g oups and examined bio ic
(endophy e s a us o he plan ,
plan o igin and plan biomass)
and abio ic (wa e and nu ien
ea men s) en i onmen al
ac o s. Signi ican en i onmen al
a iables (a) (W = wa e ,
N = ni ogen, WN = wa e and
ni ogen, C = con ol) and
plan biomass (BIOM) a e shown
wi h i e axonomical
in e eb a e g oups: he bi o es
(b), de i i o es (c), omni o es
(d), pa asi oids (e) and p eda o s
( ). Eigen alue o he i s axis
was 0.171 and o he second
axis 0.056
116 Fungal Di e si y (2011) 47:109–118
Conclusions
Ou esul s demons a e ha (1) he axonomical di e si y and
complexi y o an in e eb a e communi y can be e y high
e en in ela i ely simple plan communi ies, and (2) he
di e si y is commensu a e wi h p ima y p oduc ion and
en i onmen al ac o s ha in e ac wi h plan o igin a he
han endophy e in ec ions. Fu he mo e, in e eb a e commu-
ni y, pa icula ly he mos di e se eeding guild, he bi o es,
showed s ong di e en ia ion along he examined wa e and
nu ien g adien s. This may d i e he communi y s uc u e o
in e eb a e he bi o es in a pa chy en i onmen . The lack o
inc eased o dec eased he bi o e esis ance migh be pa ly
explained by he ac ha alkaloids in na i e Eu opean all
escue a e no o he ype o le el ha educe (A khami and
Rudge s 2009) o p omo e (Fae h and Shocha 2010;Janie
al. 2010) plan eeding in e eb a es. Howe e , such di e -
ences in alkaloid p o iles and o he plan cha ac e is ics due
o di e ences among plan o endophy e geno ypes ails o
explain he lack o axon, eeding guild and communi y le el
esponses wi h he cul i a K-31.
We p opose ha empi ical whole-communi y app oaches
a e equi ed o unde s and he impo ance o endophy es and
o he mechanisms d i ing plan popula ions and in e eb a e
communi ies eeding on hem. Accumula ing e idence om
endophy e media ed in e ac ions has e ealed ha endophy es
can nega i ely a ec plan eeding he bi o es (Saikkonen e
al. 2010). Howe e , he accumula ing e idence also indica es
ha di e si y in esul s and in e p e a ions o he gene al
impo ance o endophy es in g assland communi ies
inc eases as new model sys ems appea . Cu en li e a u e
appea s o be s ongly biased by wo model species, all
escue and pe ennial yeg ass and hei ew cul i a s such as
K-31, in in oduced and ag onomic en i onmen s, and his
has dis ac ed he li e a u e (Saikkonen e al. 2006,2010). By
using wild all escues in hei na i e con inen , we we e able
o show ha en i onmen al condi ions and hos plan o igin
o e ide endophy e e ec s on in e eb a e di e si y, com-
muni y s uc u e, and eeding guilds.
Acknowledgemen s This s udy was unded by he Academy o
Finland (P ojec no. 110658).
Open Access This a icle is dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion Noncomme cial License which pe mi s any
noncomme cial use, dis ibu ion, and ep oduc ion in any medium,
p o ided he o iginal au ho (s) and sou ce a e c edi ed.
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