RESEARCH ARTICLE
Geog aphic Va ia ion in Fes uca ub a L.
Ploidy Le els and Sys emic Fungal
Endophy e F equencies
Se da Di ihan
1
, Ma jo Helande
1,2
, Hen y Va
¨ e
3
, Ped o E. Gundel
4
, Lucas A. Ga ibaldi
5
,
J. Gonzalo N. I isa i
4
, I ma Saloniemi
1
, Ka i Saikkonen
2
1Depa men o Biology, Uni e si y o Tu ku, Tu ku, Finland, 2Na u al Resou ces Ins i u e Finland (Luke),
Tu ku, Finland, 3Bo anical Museum, Finnish Museum o Na u al His o y, Uni e si y o Helsinki, Helsinki,
Finland, 4IFEVA, Facul ad de Ag onomı
´a, Uni e sidad de Buenos Ai es, CONICET, Buenos Ai es,
A gen ina, 5G upo de In es igacio
´n en Ag oecologı´a (AGRECO), Sede Andina, Uni e sidad Nacional de
Rı
´o Neg o (UNRN) and Consejo Nacional de In es igaciones Cien ı
´ icas y Te
´cnicas (CONICET), San Ca los
de Ba iloche, Rı
´o Neg o, A gen ina
*[email p o ec ed]
Abs ac
Polyploidy and symbio ic Epichloe
¨ ungal endophy es a e common and he i able cha ac e -
is ics ha can acili a e en i onmen al ange expansion in g asses. He e we examined geo-
g aphic pa e ns o polyploidy and he equency o ungal endophy e colonized plan s in 29
Fes uca ub a L. popula ions om eigh geog aphic si es ac oss la i udes om Spain o
no he nmos Finland and G eenland. Ploidy seemed o be posi i ely and nega i ely co e-
la ed wi h la i ude and p oduc i i y, espec i ely. Howe e , he co ela ions we e nonlinea ;
84% o he plan s we e hexaploids (2n = 6x = 42), and he posi i e co ela ion be ween
ploidy le el and la i ude is he esul o only ou popula ions skewing he da a. In he sou h-
e nmos end o he g adien 86% o he plan s we e e aploids (2n = 4x = 28), whe eas in
he no he nmos end o he g adien one popula ion had only oc oploid plan s (2n = 8x = 56).
Endophy es we e de ec ed in 22 ou o he 29 popula ions. Endophy e equencies a ied
among geog aphic si es, and popula ions and habi a s wi hin geog aphic si es i espec i e
o ploidy, la i ude o p oduc i i y. The highes o e all endophy e equencies we e ound in
he sou he nmos end o he g adien , Spain, whe e 69% o plan s ha bo ed endophy es. In
no he n Finland, endophy es we e de ec ed in 30% o g asses bu endophy e equencies
a ied among popula ions om 0% o 75%, being highe in meadows compa ed o i e -
banks. The endophy es we e de ec ed in 36%, 30% and 27% o he plan s in Fa oe Islands,
Iceland and Swi ze land, espec i ely. P ac ically all examined plan s collec ed om sou h-
e n Finland and G eenland we e endophy e- ee, whe eas in o he geog aphic si es endo-
phy e equencies we e highly a iable among popula ions. Common o all popula ions wi h
high endophy e equencies is hea y e eb a e g azing. We p opose ha he de ec ed
endophy e equencies and ploidy le els mi o pas dis ibu ion his o y o F. ub a a e he
las glacia ion pe iod, and local adap a ions o pas o p e ailing selec ion o ces such as
e eb a e g azing.
PLOS ONE | DOI:10.1371/jou nal.pone.0166264 No embe 15, 2016 1 / 16
a11111
23(1 $&&(66
Ci a ion: Di ihan S, Helande M, Va¨ e H, Gundel
PE, Ga ibaldi LA, I isa i JGN, e al. (2016)
Geog aphic Va ia ion in Fes uca ub a L. Ploidy
Le els and Sys emic Fungal Endophy e
F equencies. PLoS ONE 11(11): e0166264.
doi:10.1371/jou nal.pone.0166264
Edi o : Tzen-Yuh Chiang, Na ional Cheng Kung
Uni e si y, TAIWAN
Recei ed: Augus 5, 2016
Accep ed: Oc obe 25, 2016
Published: No embe 15, 2016
Copy igh : 2016 Di ihan e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape and i s Suppo ing In o ma ion
iles.
Funding: This wo k was suppo ed by he
Academy o Finland g an s 137909, 281354 and
292732 Fund ecei e au ho : KS, as well as
INTERACT (G an Ag eemen No. 262693) unde
he Eu opean Communi y’s Se en h F amewo k
P og amme.
Compe ing In e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
In oduc ion
Biogeog aphic gene aliza ions on he ac o s esponsible o pa e ns o species’ anges a e
la gely based on compa isons o closely ela ed species [1]. Fo example, polyploidy appea s o
be posi i ely associa ed wi h la i ude, al i ude and ecen deglacia ions [2–5], and bio ic in e -
ac ions ha e been connec ed o adap i e adia ion o plan s [6–8]. Al hough di e en ploidy
le els ha e commonly been documen ed wi hin species as well, ex ensi e s udies on geog aphic
species-speci icploidy dis ibu ions and impo ance o bio ic in e ac ions a e mos ly lacking
[9], and spo adic indings a e o en con lic ing.
G asses a e a pe ec model o s udies on geog aphic ploidy dis ibu ions and impo ance
o bio ic in e ac ions because hey co e highe a ea o land han any o he g oup o plan s
ac oss all he con inen s excep An a c ica [10]. We selec ed ed escue (Fes uca ub a L. sensu
la o) as a model species o ou s udy. Fi s , i is a wild pe ennial Eu asian g ass widely dis ib-
u ed and pheno ypically a iable in he No he n hemisphe e. Plan s alling in o mo phologi-
cally dis inguishable ca ego ies a e o en inconsis en ly classi ied as bo h species and
subspecies showing local adap a ions [11,12]. A ailable li e a u e sugges s ex ensi e hyb idiza-
ion be ween (sub)species, po en ially esul ing in obse ed na u al polyploids (2n = 14, 21, 28,
42, 49, 56, 64 and 70) [11,12]. In e e ile plan s exhibi s iking mo phological a ia ion and
pa o he eco ypic di e si y is sugges ed o be ela ed o he polyploidiza ion [13]. Second, ed
escue is well known o i s a iable and occasionally high equencies o sys emic ungal endo-
phy es [14–17]–plan associa ed ungi ha a e sugges ed o ac as de ensi e plan mu ualis s
and he eby expand dis ibu ion ange o he hos g ass [7,18–21].
Bo h polyploidy and sys emic, e ically in ge mline ansmi ed Epichloë endophy es a e
common g ass cha ac e is ics ha can be adap i e o a ious en i onmen al condi ions [2,4,6,
7,22–24]. Polyploidy, he mul iplica ion o he comple e se o ch omosomes, can bes ow
adap i e po en ial and e olu iona y lexibili y on plan s and he eby imp o e hei compe i i e
and in asi e capaci y in o no he n la i udes [4,23,25]. Possessing mo e han wo se s o ch o-
mosomes can cause he e osis, shield polyploids om dele e ious e ec s o mu a ions o exam-
ple by educing he incidence o homozygous ecessi es, and bu e agains inb eeding
dep ession and gene ic d i [2,5,23]. Because polyploids a e usually unable o in e b eed wi h
hei diploid conspeci ics, polyploidy is ecognized as one o he majo mechanisms o sympa -
ic specia ion [5] and in some species he numbe o ch omosomes appea s o be posi i ely co -
ela ed wi h la i ude o al i ude [2–4,26]. Recen ly, he ole o polyploidiza ion as a modula o
o adap i e symbiosis be ween plan s and mic obes has been ecognized [27]. Fo example,
polyploidiza ion can a ec bio ic in e ac ions h ough changes in he chemical p o ile o he
plan [8]. Howe e , he empi ical e idence is a iable and pa ly con adic o y [28–30], and
he ques ion how ploidy-d i en adap a ions o en i onmen al condi ions and mic obial in e -
ac ions code e mine plan dis ibu ion is unknown.
Simila o polyploidy, Epichloë endophy es examined in his s udy can also d i e he geo-
g aphic dis ibu ion o hos g asses [31]. G ass endophy es o he genus Epichloë a e common
symbion s o cul i a ed and wild Pooideae g ass species. In he symbiosis endophy ic ungus
g ows sys emically and asymp oma ically h oughou he abo eg ound plan pa s, and
depending on he species i may be ansmi ed ei he e ically ia hos seeds and/o ho izon-
ally by sexual spo es [6,7]. In many species, he ungus is mos ly asexual and is ansmi ed
e ically om mo he plan o i s o sp ing. Ve ical ansmission is supposed o p omo e
ideli y be ween pa ne s and lead o mu ualis ic symbiosis because he i ness o he he i able
ungus and he hos g ass is igh ly linked [32–38]. Thus, he symbioses a e commonly hough
o be mu ualis ic. Nume ous s udies ha e demons a ed ha Epichloë species can inc ease
g ass esilience o d ough , looding,pa hogens and he bi o es, and hus p omo e hei
Polyploidy and Endophy ic Symbiosis ac oss La i ude
PLOS ONE | DOI:10.1371/jou nal.pone.0166264 No embe 15, 2016 2 / 16
compe i i e abili y in g ass communi ies [14,18,21,39–41]. The de ensi e mu ualism agains
he bi o es due o myco oxins appea s o p o ide he mos p e alen g ound o mu ualis ic
endophy e-g ass in e ac ions [6,7,20,21,42,43]. Howe e , an inc easing numbe o empi ical
s udies has e ealed ha he symbiosis can ange om an agonis ic o mu ualis ic in e ac ions,
and mu ualism is less equen in wild g asses compa ed o ag onomic g asses in nu ien - ich
en i onmen s [20,21,38,44,45].
This s udy aims o e eal po en ial linkages be ween geog aphic pa e ns o polyploidy and
plan - ungal endophy e symbiosis. We explo e bo h ploidy le els and endophy e equencies in
ed escues o e a wide ange o en i onmen s and la i udes ac oss Eu ope. The bene i s om
Epichloë species a e ound o be posi i ely co ela ed wi h high nu ien a ailabili y and p o-
duc i i y [20,46], and polyploidy is belie ed o inc ease owa ds less p oduc i e highe la i-
udes and al i udes [4,23]. Thus, we s udied p ima y p oduc i i y ( he no malized di e ence
ege a ion index, NDVI) o ou s udy si es [47]. We also hypo hesize ha ploidy le els and he
equencies o endophy e symbiosis would be co ela ed, because polyploidiza ion can modu-
la e plan -mic obe in e ac ions and bo h polyploidiza ion and sys emic g ass endophy es can
p omo e hos i ness. Because bene i s o endophy es a e pa icula ly ob ious in high nu ien
en i onmen s, we may assume ha he posi i e co ela ion should be s onge in en i onmen s
wi h he highes p ima y p oduc ion. This assump ion is suppo ed by a ecen b oad-scale
s udy [46] sugges ing ha p ima y p oduc ion is posi i ely associa ed wi h he occu ence o
sys emic g ass endophy e symbiosis.
Ma e ials and Me hods
Mo e han one housand ed escue (Fes uca ub a L.) plan s in o al we e collec ed om 29
popula ions (10–70 plan s/popula ion) om eigh geog aphic si es ac oss Eu ope (Spain, Swi -
ze land, sou he n Finland, Fa oe Islands, Iceland, wo a eas in no he n Finland and G een-
land; Table 1). Plan s we e no collec ed om na ional pa ks o o he p o ec ed a eas equi ing
pe missions. In No dic coun ies “e e yman’s igh ” gi es e e yone igh s o access, enjoy o
ec ea ion and collec unp o ec ed plan s, be ies and mush ooms in a way which does no
damage he en i onmen o dis u b o he s ega dless who owns o occupies he land. Plan s
om Spain and Swi ze land we e collec ed om public land. Geog aphical coo dina es, al i-
ude om he sea le el and ea u es o he si e we e eco ded o each popula ion (Table 1).
To ensu e he p ope species iden i ica ion o he plan s and ha collec ed plan s ep esen
indi idual gene s, only lowe ing indi iduals g owing a leas 10 me e s apa om each o he
we e collec ed. Plan s we e dug up wi h a soil co e and placed in o plas ic bags o anspo a-
ion. All he collec ed g asses we e plan ed in 250 ml po s wi h added pea and sand mix
a ound he o iginal soil co e and kep in a g eenhouse in Tu ku Uni e si y Bo anical Ga den
(60°26’N, 22°10’E) in ambien dayligh and 20–24°C (summe ime) and 4–8°C (win e ime)
empe a u es.
The collec ion si es ep esen a b oad biogeog aphical egion a ying in e ms o la i udes,
al i udes, clima ic zones (con inen al, oceanic), biological selec ion p essu es such as g azing
(Table 1) and seasonal changes in abio ic en i onmen al condi ions. Fo example, si es in
Spain ep esen g assland and xe ophy ic o es , bo h in Medi e anean clima e cha ac e ized
by summe d ough s and ainy win e s. Si es loca ed on highe la i udes a e cha ac e ized by
s onge seasonal changes in day leng h and associa ed ligh quali y limi ing p ima y p oduc-
ion [48], sho g owing seasons in summe and long and cold win e s. Howe e , oceanic si es
(Iceland and Fa oe Islands) s ongly a ec ed by he Gul -s eam a e cha ac e ized by high p e-
cipi a ion yea - ound, cool summe s and ela i ely mild win e s compa ed o he o he si es on
he same la i udes.
Polyploidy and Endophy ic Symbiosis ac oss La i ude
PLOS ONE | DOI:10.1371/jou nal.pone.0166264 No embe 15, 2016 3 / 16
Table 1. The collec ion si es o Fes uca ub a plan s and hei a ibu es. N = numbe o collec ed plan s. Mean NDVI = mean no malized di e ence
ege a ion index es ima ed om yea 2000 o 2012. In . % = pe cen age o endophy e in ec ed plan s (endophy e equency) in popula ion. 4x %, 6x % and
8x % = pe cen age o e aploids, hexaploids and oc oploids, espec i ely, in popula ion.
Popula ion
code
Geog aphic
si e
Popula ion N Geog aphic
coo dina es
Al i ude
(m a.s.l.)
Mean
NDVI
In .
%
4x
%
6x
%
8x
%
Fea u es o he si e G azing
SP1 Spain Ca
´ce es 31 N 40˚12’1’’ W
5˚45’11’’
768 0,59 81 100 0 0 Xe ophy ic o es High
(ca le)
SP2 Spain Salamanca
1
27 N 40˚56’20’’ W
6˚7’6’’
863 0,57 67 78 22 0 Meadow High
(ca le)
SP3 Spain Salamanca
2
37 N 40˚58’24’’ W
5˚57’33’’
812 0,51 59 81 3 16 Meadow High
(ca le)
SW1 Swi ze land Ande ma 26 N 46˚32’19’’ E
8˚40’31’’
1500 0,42 23 0 96 4 Meadow wi h na u al
g assland ege a ion,
slope acing eas
High
(ca le)
SW2 Swi ze land Biez 25 N 46˚37’42’’ E
8˚35’26’’
1600 0,41 36 0 100 0 Meadow wi h na u al
g assland ege a ion,
slope acing no h-wes
High
(ca le)
SW3 Swi ze land Piasca 23 N 46˚53’56’’ E
8˚42’9’’
1850 0,19 22 0 100 0 Meadow wi h na u al
g assland ege a ion,
sawed e e y second yea ,
slope acing sou h
High
(ca le)
FI1 Sou he n
Finland
Hanko 1 42 N 59˚50’23’’ E
23˚13’40’’
0 0,55 0 2 98 0 Meadow along he coas Low
FI2 Sou he n
Finland
Hanko 2 44 N 59˚50’27’’ E
23˚13’15’’
0 0,53 0 0 93 7 Meadow along he coas Low
FI3 Sou he n
Finland
Hanko 3 40 N 59˚53’0’’ E
23˚5’52’’
0 0,43 0 5 95 0 Meadow along he coas Low
FO1 Fa oe Sandoy 39 N 61˚50’11’’ W
6˚51’21’’
69 0,42 21 3 97 0 Meadow High
(sheep)
FO2 Fa oe Nolsoy 41 N 62˚1’15’’ W
6˚41’8’’
55 0,33 5 0 98 2 Meadow High
(sheep)
FO3 Fa oe Mykines 37 N 62˚5’51’’ W
7˚40’56’’
125 0,16 68 0 100 0 Meadow High
(sheep)
FO4 Fa oe Vaga 24 N 62˚6’59’’ W
7˚26’43’’
246 0,30 25 0 83 17 Meadow High
(sheep)
FO5 Fa oe Eys u oy 39 N 62˚17’24’’ W
7˚2’10’’
316 0,34 54 5 87 8 Meadow High
(sheep)
FO6 Fa oe Vidoy 32 N 62˚22’3’’ W
6˚32’32’’
148 0,31 44 9 91 0 Meadow High
(sheep)
IC1 Iceland Iceland 1 44 N 64˚47’34’’ W
21˚32’0’’
390 0,28 32 2 98 0 Meadow High
(sheep)
IC2 Iceland Iceland 2 34 N 64˚48’52’’ W
23˚23’14’’
10 0,34 32 0 97 3 Meadow High
(sheep)
IC3 Iceland Iceland 3 42 N 66˚1’21’’ W
20˚23’39’’
38 0,30 26 5 95 2 Meadow High
(sheep)
GR1 G eenland G eenland
1
70 N 69˚14’59’’ W
53˚31’15’’
0 0,16 3 0 100 0 Meadow along he coas Low
GR2 G eenland G eenland
2
10 N 69˚15’27’’ W
53˚32’40’’
0 0,19 0 0 100 0 Meadow along he coas Low
FI4 No he n
Finland 1
Hal i 1 22 N 69˚15’0’’ E
21˚24’36’’
860 0,20 0 0 100 0 Meadows along i ule s
abo e ee-line wi h
pa chy g ass and sedge
domina ed ege a ion
Mode a e
( eindee )
FI5 No he n
Finland 1
Hal i 2 42 N 69˚15’0’’ E
21˚19’12’’
900 0,11 0 0 0 100 Mode a e
( eindee )
FI6 No he n
Finland 1
Hal i 3 32 N 69˚16’12’’ E
21˚19’12’’
920 0,28 0 0 100 0 Mode a e
( eindee )
FI7 No he n
Finland 2
Ke o 1 34 N 69˚38’6’’ E
27˚5’1’’
91 0,31 56 3 97 0 Meadow High
( eindee )
(Con inued)
Polyploidy and Endophy ic Symbiosis ac oss La i ude
PLOS ONE | DOI:10.1371/jou nal.pone.0166264 No embe 15, 2016 4 / 16
Ploidy de e mina ion
Ploidy le els o he plan s we e de e mined by low cy ome y(FCM) [49]. We used known
ch omosome coun s o F. ub a plan s as e e ences o he FCM esul s [49]. Plan s om di -
e en popula ions (one o h ee plan s o each popula ion) we e andomly chosen o mic o-
scopically de e mine cy o ype. These e e ence plan s we e g own hyd oponically o p oduce
esh oo ips. Asep ically cu oo ips we e p e ea ed wi h 1% 1-alphab omonaph alene and
s ained in 2% ace ic o cein solu ion [50]. The oo ips we e hen squashed in a d op o 45%
ace ic acid on he slides and analyzed unde mic oscope. P epa a ions we e moun ed wi h
en halan a e he me aphases we e pho og aphed.
We sampled a ca. 0.5 cm
2
lea piece om each plan . The sample was chopped in a glass
Pe i dish wi h an asep ic azo blade in 1 ml ice-cold nuclei isola ion bu e (LBO1 in one-s ep
p ocedu e). The suspension was mixed by sucking and discha ging wi h he pipe e se e al
imes and hen il e ed in o an Eppendo ube using a 50 μm nylon mesh. DNA luo och ome
s ock solu ion wi h 50 μl ml
-1
p opidium iodide (PI) and 50 μl ml
-1
ibonuclease (RNase) was
added and incuba ed on ice one hou be o e FCM analysis [51]. The 96 well pla e-based FCM
p ocedu e [49] was ca ied ou using LSR II (Bech on Dickinson San Jose, USA) low cy ome-
e a he Tu ku Cen e o Bio echnology, Finland. Pisum sa i um L. ‘C i ad’ (2C DNA
alue = 9.09 pg) plan s ob ained om he Ins i u e o Expe imen al Bo any (Labo a o y o
Molecula Cy ogene ics and Cy ome y, Olomouc, Czech Republic), we e used as an ex e nal
e e ence o de e mine DNA quan i y in pic og ams (pg). In addi ion, known e aploid and
hexaploid F. ub a plan s we e used o each FCM un. The FCM channel was de e mined as
G
1
peak o each sample and DNA ploidy le els we e es ima ed as ollows:
Ploidy le el o sample
¼G1peak lou escence o sample ðmedianÞ  G1peak lou escence o e e ence ðmedianÞ
Ploidy le el o e e ence
The low cy ome ic da a we e measu ed wi h Flowing So wa e e sion 2.4.1 (Pe u Te ho,
Tu ku Cen e o Bio echnology, Finland; www. lowingso wa e.com).
Endophy e de ec ion
Fungal endophy e s a us (endophy e in ec ed E+ / endophy e ee E-) o each o he s udy
plan s was de ec ed by pla ing h ee lea shea hs ɖsu ace- s e ilized by incuba ion o 1 min in
90% e hanol, 4 min in 4% sodium hypochlo i e and 30 s in 90% e hanol ɖcu in o 5 pieces and
placed on po a o dex ose aga (5% PDA). The Pe i dishes we e moni o ed up o h ee weeks
Table 1. (Con inued)
Popula ion
code
Geog aphic
si e
Popula ion N Geog aphic
coo dina es
Al i ude
(m a.s.l.)
Mean
NDVI
In .
%
4x
%
6x
%
8x
%
Fea u es o he si e G azing
FI8 No he n
Finland 2
Ke o 2 40 N 69˚43’56’’ E
27˚12’0’’
85 0,31 75 3 85 13 Meadow High
( eindee )
FI9 No he n
Finland 2
Ke o 3 34 N 69˚45’32’’ E
26˚59’19’’
107 0,31 50 0 94 6 Meadow High
( eindee )
FI10 No he n
Finland 2
Ke o 4 42 N 69˚54’36’’ E
27˚1’48’’
73 0,27 45 2 98 0 Ri e bank High
( eindee )
FI11 No he n
Finland 2
Ke o 5 31 N 69˚56’11’’ E
26˚27’45’’
106 0,28 23 0 94 6 Ri e bank High
( eindee )
FI12 No he n
Finland 2
Ke o 6 35 N 69˚56’41’’ E
26˚43’22’’
85 0,29 20 0 100 0 Ri e bank High
( eindee )
doi:10.1371/jou nal.pone.0166264. 001
Polyploidy and Endophy ic Symbiosis ac oss La i ude
PLOS ONE | DOI:10.1371/jou nal.pone.0166264 No embe 15, 2016 5 / 16

o hei sys emic endophy e ungal g ow h. When ypical whi e Epichloë ungal endophy e
mycelia g ew ou om se e al lea pieces, he plan was conside ed as endophy e in ec ed [16].
The in ec ion s a us o indi idual plan s was e i ied la e by s aining and mic oscopic exami-
na ion o se e al seeds o each plan . Sys emic and e ically ia hos g ass ansmi ed endo-
phy ic ungi a e hos species-speci ic.Simila ly o o he s udies, we ha e iden i ied he ungus
associa ed wi h ed escue as E. es ucae by compa ing he DNA sequences wi h Blas sea ches
o GeneBank in ou p e ious s udies [16].
NDVI
To compa e ege a ion p oduc i i y o he s udy si es quan i a i ely, we calcula ed he no mal-
ized di e ence ege a ion index (NDVI) o each popula ion sepa a ely using NASA MODIS
sa elli e images (Fig 1,Table 1). The NDVI accu a ely es ima es unc ional a ibu es o he eco-
sys em such as abo eg ound ne p ima y p oduc ion (ANPP), i s in e -annual a ia ion and
ege a ion phenology [47,52,53]. NDVI is closely and posi i ely co ela ed wi h lea a ea and
he ac ion o pho osyn he ically ac i e adia ion abso bed by g een ege a ion [54–56]. NDVI
da a uniquely allows si e cha ac e iza ion because i ep esen s he speci ic consequences o
en i onmen al and human e ec s on ege a ion unc ioning.
We ob ained NDVI alues om he MODIS p ojec h ough he MODIS global subse s ool
(h p://daac.o nl.go /cgibin/MODIS/GLBVZ1Glb/modissubse o de globalcol5.pl). We used
he MOD 13 Vege a ion Indices p oduc , g idded, 16-day composi e images wi h 250-m pixel
size. We ex ac ed wo essen ial a ibu es o abo eg ound p ima y p oduc ion dynamics by
calcula ing he NDVI a e age annual in eg al and i s in e -annual coe icien o a ia ion om
Feb ua y 2000 o Decembe 2012 (Fig 1). These ai s a e known o cap u e impo an ea u es
o ecosys em unc ioning [57].
S a is ical analysis
We used linea models (linea eg ession) o analyse he e ec s o mean NDVI and la i ude on
endophy e in ec ion equencies and mean ploidy o popula ions. No mali y o esiduals was
checked g aphically and using Kolmogo o -Smi no es esul s o he models.
We used a logis ic eg ession model o es ima e ploidy le el, al i ude (Al ), la i ude (La ) and
hei pai -wise in e ac ions as ixed e ec s on endophy e s a us ( wo le els: E+ and E-) using
binomial e o dis ibu ion (logi link). We also included popula ion as a andom e ec ( an-
dom in e cep model) o accoun o he ac ha indi idual plan s we e spa ially nes ed wi hin
popula ions (29 popula ions in o al). We used AIC (Akaike In o ma ion C i e ion) o selec
bes - i ing models o all combina ions o ixed-e ec a iables. AIC alues we e ob ained
based on maximum-likelihoodes ima es o eg ession coe icien s,because models di e ed in
hei ixed s uc u e bu sha ed he same andom s uc u e ( andom in e cep s), whe eas
pa ame e es ima es o inal models p esen ed in igu es we e ob ained using he es ic ed
maximum likelihood me hod [58]. Models we e es ima ed using lme unc ion o he lme4
package [59] in he R so wa e [60], and AIC and Akaike’s weigh o each model o all possible
models based on di e en combina ions o he p edic o a iables we e ob ained wi h he
d edge unc ion o he MuMln package [61] in he R so wa e.
Resul s
O e all 84%, 9% and 7% o he plan s we e hexaploids (2n = 6x = 42), e aploids (2n = 4x =
28) and oc oploids (2n = 8x = 56), espec i ely. Mo e han one ploidy le el was de ec ed in 19
ou o 29 popula ions (Table 1). Ploidy le el seems o be pa ly linked wi h p oduc i i y and la -
i ude; ploidy posi i ely and nega i ely associa ed wi h la i ude (p<0.0001) and p oduc i i y
Polyploidy and Endophy ic Symbiosis ac oss La i ude
PLOS ONE | DOI:10.1371/jou nal.pone.0166264 No embe 15, 2016 6 / 16
(p<0.0001), espec i ely (Fig 2). I is no ewo hy, howe e , ha majo i y o he plan s we e
hexaploids (2n = 6x = 42), and he posi i e co ela ion be ween ploidy le el and la i ude was
no linea , and he co ela ion is due o he in luence o ou de ian popula ions. In he sou h-
e nmos geog aphic si e, Spain, 86% o he plan s we e e aploids (2n = 4x = 28), and in no h-
e nmos Finland all o he plan s in one popula ion a high al i ude (in Hal i, 900m) we e
Fig 1. Mon hly mean no malized di e ence ege a ion index (mean NDVI) alues o he Fes uca ub a collec ion
si es ( o popula ion codes see Table 1) om Feb ua y 2000 o Decembe 2012.
doi:10.1371/jou nal.pone.0166264.g001
Polyploidy and Endophy ic Symbiosis ac oss La i ude
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oc oploids (2n = 8x = 56) (Table 1,Fig 2). Ou side hese ex emes o he la i udinal ange, hexa-
ploid plan s we e dominan . In G eenland, all he plan s we e hexaploids, and in Swi ze land
98% o he plan s we e hexaploids and only 2% oc oploids (Table 1). In he o he popula ions
e aploids and oc oploids we e spo adically dis ibu ed (Table 1).
Endophy e in ec ions we e de ec ed in 22 ou o 29 ed escue popula ions (Fig 3). The
se en o ally endophy e- ee popula ions we e he h ee popula ions om Hanko in sou he n
Finland, he h ee popula ions om Hal i in no he n Finland and one popula ion om G een-
land. Fu he mo e, only one in ec ed g ass was ound in he o he popula ion in G eenland
(Table 1,Fig 3). O e all in ec ion equency o he s udy a ea was 29% bu equencies a ied
i espec i e o ploidy (p = 0.14) (Table 2) among geog aphic si es, popula ions wi hin geo-
g aphic si es and among habi a s (Fig 3,Table 1).
The highes o e all in ec ion equencies we e ound in Spain (69%) whe e occu ence o
in ec ions was high in all popula ions (Fig 3). A Ke o in no he n Finland, on a e age 45% o
g asses we e in ec ed bu in ec ion equencies we e much highe in meadows (60%) compa ed
o nea by i e banks (29%). The occu ence o in ec ions in Fa oe Islands, Iceland and Swi ze -
land we e 36%, 30% and 27%, espec i ely. In hese geog aphic si es a ia ion in endophy e
occu ence among popula ions was conside able only in Fa oe Islands a ying om 5% o 68%
(Table 1,Fig 3).
La i ude appea s no o be linked o he de ec ed a ia ion in endophy e occu ence
(p = 0.65) (Table 2,Fig 4). Ins ead, in ec ion equencies appea o be associa ed wi h al i ude
(p = 0.04) bu in e ac i ely wi h la i ude (p = 0.03) (Table 2). The sampling was no , howe e ,
designed o es he impo ance o al i ude and hus, hese esul s emain inconclusi e. In pop-
ula ions collec ed om low la i udes, in Spain and Swi ze land, he endophy e equencies we e
lowes in Swi ze land whe e all collec ed popula ions we e om high al i ude (Table 1). In con-
as , elsewhe e in ec ion equencies a ied i espec i e o al i ude. Fo example, popula ions
collec ed om G eenland and Hanko, bo h si ua ed a sea le el, and Hal i si ua ed a 900 m
abo e sea le el we e endophy e- ee (Table 1) sugges ing he al i ude canno accoun o pa -
e ns o endophy e occu ence.
Endophy e equencies o he g ass popula ions we e no associa ed wi h p oduc i i y (p = 0.16)
(Fig 5). No malized di e ence ege a ion index (NDVI) alues a ied among geog aphic si es
a he han along la i ude (Table 1,Fig 6). The highes geog aphic si e speci icmean-NDVI alues
Fig 2. The e ec o la i ude (a) and mean no malized di e ence ege a ion index (mean NDVI) (b) on mean ploidy o Fes uca ub a popula ions.
Fi ed eg ession line in (a) is o illus a i e pu pose since s a is ical es s a e sugges i e due o p oblems o no mali y in he da a.
doi:10.1371/jou nal.pone.0166264.g002
Polyploidy and Endophy ic Symbiosis ac oss La i ude
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0.51, 0.56 and 0.31 we e es ima ed o sou he n Finland, Spain and Iceland-Fa oe Islands, espec-
i ely (Table 1). Co esponding endophy e in ec ion equencies we e 0% (sou he n Finland), 69%
(Spain), 30% (Iceland) and 36% (Fa oe Islands) demons a ing ha o e all p oduc i i y o he geo-
g aphic si e is unlikely o be linked wi h endophy e in ec ion equencies in ed escue popula ions.
Mon hly mean NDVI es ima es, howe e , clea ly demons a e ha p ima y p oduc ion is season-
ally limi ed in all he o he s udy si es excep in Spain (Fig 1).
Discussion
Ou esul s do no suppo he hypo heses ha polyploidiza ion and he occu ence o sys emic
ungal endophy es in ed escue show la i udinal g adien s, o ha hey a e co ela ed wi h
Fig 3. Collec ion si es and endophy e in ec ion equencies o Fes uca ub a popula ions. In ec ion equency ci cles a e shown wi h he
popula ion codes (Table 1).
doi:10.1371/jou nal.pone.0166264.g003
Table 2. E ec s o ploidy, la i ude, al i ude and in e ac ion be ween la i ude and al i ude (La x Al ) on endophy e s a us o Fes uca ub a plan s.
Es ima e S d. E o z alue P (!|z|)
In e cep -4.78 5.08 0.94 0.35
Ploidy 0.03 0.02 1.48 0.14
La i ude 0.04 0.08 0.44 0.65
Al i ude 0.01 0.00 2.03 0.04*
La x Al -0.00 0.00 -2.14 0.03*
*, p0.05
**, p0.01
***, p0.001
doi:10.1371/jou nal.pone.0166264. 002
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