RESEARCH ARTICLE
Seasonal Succession o Fungi Associa ed wi h
Ips ypog aphus Bee les and Thei Pho e ic
Mi es in an Ou b eak Region o Finland
Riikka Linnakoski
1,2
*, Saila Mahilainen
3
, Alison Ha ing on
4
, Hen i Vanhanen
5
,
Miikka E iksson
6
, Lau i Meh ä alo
7
, A i Pappinen
3
, Michael J. Wing ield
2
1Depa men o Fo es Sciences, Facul y o Ag icul u e and Fo es y, Uni e si y o Helsinki, Helsinki,
Finland, 2Depa men o Mic obiology and Plan Pa hology, Fo es y and Ag icul u al Bio echnology Ins i u e
(FABI), Uni e si y o P e o ia, P e o ia, Sou h A ica, 3School o Fo es Sciences, Facul y o Science and
Fo es y, Uni e si y o Eas e n Finland, Joensuu, Finland, 4Wa son Founda ion, New Yo k, Uni ed S a es o
Ame ica, 5Na u al Resou ces Ins i u e Finland (Luke), Joensuu, Finland, 6School o Applied Educa ional
Science and Teache Educa ion, Philosophical Facul y, Uni e si y o Eas e n Finland, Sa onlinna, Finland,
7School o Compu ing, Uni e si y o Eas e n Finland, Joensuu, Finland
* iikka.linnakoski@helsinki. i
Abs ac
The ophios oma oid ungi (Mic oascales and Ophios oma ales, Ascomyco a) a e common
associa es o Ips ypog aphus, and include ee pa hogens and species esponsible o
blue-s ain o imbe . Fungal assemblages associa ed wi h I. ypog aphus ha e a ied con-
side ably be ween s udies bu ew in es iga ions ha e a emp ed o explain his a ia ion.
Fo his eason, we assessed he o e all cul i able ungal di e si y associa ed wi h I. ypo-
g aphus in a s o m- elled sp uce o es in sou h-eas e n Finland. Fungi we e isola ed om
he indi idually collec ed bee les as well as hei pho e ic mi es in sp ing, summe and
au umn, including di e en li e s ages o he bee le (hibe na ion, dispe sal ligh and i s
gene a ion). The in e nal ansc ibed space (ITS) gene egion was used o iden i y he
ungi. A o al o 32 ope a ional axonomic uni s (OTUs) we e ound and hese esided in ou
ungal phyla/subphyla (24 Ascomyco a, 2 Basidiomyco a, 5 Muco omyco ina, 1 Mo ie ello-
myco ina) in associa ion wi h adul ba k bee les. Ophios oma oid species we e he mos
commonly de ec ed ungal associa es. A gene alized linea model analysis showed a clea
associa ion be ween ungal communi ies and season, indica ing seasonal succession
among I. ypog aphus-associa ed ungi. The season o sampling appea s o be an impo an
ac o ha has esul ed in inconsis encies be ween esul s in p e ious s udies. Many o
hese ungi we e also ound on pho e ic mi es and hei p esence o absence could ha e
in luenced a ia ion in pa e ns o associa ion.
In oduc ion
The Eu opean sp uce ba k bee le (Ips ypog aphus) is he mos economically and ecologically
impo an ba k bee le species in coni e ous o es s o Eu ope. I in es s mainly wind- elled and
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 1/14
a11111
OPEN ACCESS
Ci a ion: Linnakoski R, Mahilainen S, Ha ing on A,
Vanhanen H, E iksson M, Meh ä alo L, e al. (2016)
Seasonal Succession o Fungi Associa ed wi h Ips
ypog aphus Bee les and Thei Pho e ic Mi es in an
Ou b eak Region o Finland. PLoS ONE 11(5):
e0155622. doi:10.1371/jou nal.pone.0155622
Edi o : Olle Te enius, Swedish Uni e si y o
Ag icul u al Sciences, SWEDEN
Recei ed: Janua y 25, 2016
Accep ed: May 2, 2016
Published: May 17, 2016
Copy igh : © 2016 Linnakoski 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 : The sequence da a
gene a ed in his s udy a e a ailable in GenBank
da abase (accession numbe s KT896627-
KT896658). Rep esen a i e isola es o
ophios oma oid ungi a e p ese ed in he Cul u e
Collec ion (CMW) o he Fo es y and Ag icul u al
Bio echnology Ins i u e (FABI), Uni e si y o P e o ia,
Sou h A ica.
Funding: The Uni e si y o Helsinki, h ps://www.
helsinki. i/en (RL); Jouko Tuo olan Sää iö (SM); The
Ol i Founda ion, h p://www.ol isaa io. i/web/
ol isaa io/ (SM); The Thomas J. Wa son Fellowship
weakened ees and is capable o killing heal hy ees in la ge numbe s only when he popula-
ion exceeds ce ain dange ous le els. Ips ypog aphus is one o he d i ing o ces o o es suc-
cession and, he e o e, an impo an componen o na u al o es ecosys ems [1]. Howe e ,
mass ou b eaks o he pes esul ing in se ious losses o o es y ha e mo e equen ly been
epo ed in No he n Eu ope. I has been sugges ed ha clima e change migh al e bo h he
equency and he in ensi y o o es pes ou b eaks [2–4]. Se e i y o I. ypog aphus ou b eaks
has been linked o d y summe s combined wi h wa me empe a u es [4]. In his ega d, cli-
ma e change ollowed by inc easing empe a u es du ing he b eeding season could di ec ly
a ec he popula ion dynamics o I. ypog aphus, allowing he ba k bee le o comple e wo
a he han he no mal one gene a ion pe yea in No he n Eu ope [5]. Inc eased numbe s o
s o ms and d ough pe iods associa ed wi h clima e change will also indi ec ly inc ease he isk
o ba k bee le ou b eaks ia changes in a ailabili y o sui able b eeding ma e ial [6].
Du ing ou b eaks, I. ypog aphus colonizes ees ia phe omone-media ed mass a acks and
in conce wi h i s ungal associa es. This symbiosis has been hypo hesized o exhaus hos ee
de enses [7–8]. Howe e , ba k bee les also ec o ungi in non-ou b eak condi ions, and he
posi i e ole o he ungal associa es in he ee-killing by ba k bee les has been challenged [9].
Nume ous s udies ha e been conduc ed on he ungi associa ed wi h I. ypog aphus, and pa ic-
ula ly he biodi e si y o ophios oma oid [10] ungi belonging o Mic oascales and Ophios o-
ma ales (Ascomyco a) in No he n Eu ope (e.g. [11–23]) and Geosmi hia ungi in Cen al and
No heas e n Eu ope [24–25]. While ungal assemblages associa ed wi h his ba k bee le spe-
cies ha e a ied conside ably be ween s udies, only a ew in es iga ions ha e a emp ed o
explain his a ia ion [17–18,26–27].
The aim o he p esen s udy was o conside he seasonal a ia ion in ungal assemblages
associa ed wi h di e en s ages o I. ypog aphus in Finland. The o e all ungal di e si y associ-
a ed wi h I. ypog aphus including i s pho e ic mi es in a s o m- elled sp uce o es in sou h-
eas e n Finland was in es iga ed.
Ma e ials and Me hods
S udy a ea and collec ion o ba k bee les and mi es
In July and Augus 2010, o e 8 million m
3
o ee imbe was damaged in sou he n and middle
Finland hunde s o ms. Ou s udy a ea was loca ed in one o he mos se e ely damaged o es s
in Ruokolah i, sou h-eas e n Finland (61° 492’N, 29° 054’E). The su ounding s and is mainly
My illus ype [28] wi h ma u e coni e s domina ed by Picea abies, and pa ly by Pinus syl es-
is. The s o m- elled ees comp ised old-g ow h logs and he e was a high olume o decaying
wood p esen in he egion. Fo hese easons sal age logging and emo al o he s o m- elled
ees was di icul o conduc . The allen ees we e consequen ly le in he o es and a p i a e
74 hec a e o es p ese e was es ablished in 2010 in he mos se e ely damaged egion in Vii a-
lampi, Ruokolah i. I was ob ious ha la ge olumes o sui able b eeding ma e ial would esul
in an I. ypog aphus ou b eak in he egion o e he ollowing yea s, especially whe e a o able
wea he condi ions p e ailed. The I. ypog aphus popula ion was moni o ed using phe omone
ap ca ches and an assessmen o bee le-killed ees. Sp ead o he ba k bee le ou b eak was
con olled by sani a ion logging in he su ounding a eas. Ba k bee les we e collec ed adjacen
o he newly es ablished na u e ese e a ea du ing 2013 o which he To na o Oyj issued a
esea ch pe mi .
In Ap il 2013, o e win e ing I. ypog aphus adul s we e collec ed om he o es li e and
unde he ba k o s o m- elled ees. Each bee le hibe na ing unde he ba k was collec ed om
a sepa a e galle y. Samples o o es li e we e collec ed app oxima ely 0.5–1.0 m dis ance
om he ee base, and 1–24 bee les we e ound om each li e sample. To a oid possible
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 2/14
g an ed by he Wa son Founda ion, h p://wa son.
ounda ion (AH); The Na u al Resou ces Ins i u e
Finland (Luke), h ps://www.luke. i/en (HV); The
Uni e si y o Eas e n Finland, h p://www.ue . i/en
(ME, LM, AP); The Uni e si y o P e o ia, h p://www.
up.ac.za (RL, MJW); The membe s o he T ee
P o ec ion Co-ope a i e P og amme (TPCP) and he
THRIP ini ia i e o he Depa men o T ade and
Indus y, Sou h A ica (RL, MJW). 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 .
c oss-con amina ion o he samples, all he ba k bee les we e collec ed indi idually wi h s e ile
o ceps. Only li ing bee les we e p ocessed and each bee le was indi idually placed in o a s e ile
1.5 ml Eppendo ube. Du ing he dispe sal ligh pe iod in ea ly June 2013, ba k bee les we e
lu ed o he ou e su ace o he unks o he ap ees by using Ipsowi
1
S anda d (Wi asek,
P lanzenSchu z GmbH, Aus ia) phe omone s ips. The i s gene a ion adul s we e collec ed
a he end o Oc obe and ea ly No embe om unde he ba k o s o m- elled ees as well as
newly a acked s anding ees, and om he o es li e .
The bee les we e anspo ed o he labo a o y and s o ed a -20°C o 24 hou s. A ba k bee-
le species mo phologically simila o I. ypog aphus,Ips ami inus, was also p esen in he s ud-
ied egion. The e o e, a e eeze- ea men , he iden i ica ion o each bee le was con i med
unde dissec ion mic oscope and I.ami inus indi iduals we e excluded om his s udy. A he
same ime, all pho e ic mi es p esen on he bee les we e collec ed indi idually and ans e ed
o new s e ile Eppendo ubes. We did no a emp o iden i y he mi es. The ubes con aining
bee les and mi es we e s o ed a 5°C and ungal isola ions we e made om hem on he same
day ha hey we e iden i ied.
Isola ion and mo phological g ouping o ungi
Each ba k bee le and pho e ic mi e was indi idually c ushed and placed on o he su ace o
Mal Ex ac Aga (MEA; 2% mal ex ac om Bioka Diagnos ics, Beau ais, F ance and 2%
aga om Fishe Scien i ic, Mexico) in Pe i dishes con aining 0.05 g/l o s ep omycin sulpha e
(Sigma-Ald ich, China). The pla es we e hen incuba ed a 25°C o 2–4 weeks and obse ed
daily o ungal g ow h. When ungal g ow h was obse ed, spo e masses and/o ungal mycelia
we e ans e ed o esh 2% MEA pla es (wi hou an ibio ics) and sub-cul u ed un il pu e cul-
u es we e ob ained. Pu i ied ungal isola es we e hen examined unde dissec ion mic oscope
and g ouped based on hei colony cha ac e is ics. Depending on he size o he mo phological
g oup, 1–4 isola es om each g oup we e chosen o iden i ica ion based on DNA sequence
compa isons. Rep esen a i e isola es o ophios oma oid ungi we e deposi ed in he cul u e
collec ion (CMW) o he Fo es y and Ag icul u al Bio echnology Ins i u e (FABI), Uni e si y
o P e o ia, Sou h A ica.
DNA ex ac ion, ampli ica ion and sequencing
Pu i ied ungal isola es we e g own on 2% MEA in 7 cm Pe i dishes a 25°C o up o 2 weeks
p io o DNA ex ac ion. Genomic DNA was ex ac ed using P epMan™Ul a Sample p epa a-
ion eagen (Applied Biosys ems, Fos e Ci y, CA, USA). The molecula ma ke used in his
s udy was he in e nal ansc ibed space (ITS) egion o he nuclea ibosomal DNA, which
has i s limi a ions bu is gene ally su icien o mos ungi a leas o he eliable iden i ica ion
a he species complex le el. The ITS gene egion was ampli ied using a p ime pai ITS1-F
[29] and ITS4 [30].
Ampli ica ion o he s udied gene egion and pu i ica ion o he PCR p oduc s we e pe -
o med using he same p o ocols desc ibed in ou p e ious publica ion [31]. The PCR eac ion
mix u e con ained 0.15 μL o MyTaq™DNA Polyme ase (5 U/μl) (Bioline, Massachuse s,
USA), 2.5 μl o MyTaq™Reac ion Bu e (5×) con aining dNTPs, MgCl
2
and enhance s o he
op imal pe o mance, and 0.50 μl o each p ime (Whi ehead Scien i ic L d, Cape Town, Sou h
A ica). PCR eac ions we e pe o med using an ABI 2720 The mal Cycle (Applied Biosys-
ems, Fos e Ci y, CA, USA) as ollows: an ini ial dena u a ion s ep a 95°C o 2 min, ollowed
by 35 cycles o 30 s a 94°C, 30 s a 55°C and 1 min a 72°C, and a inal chain elonga ion a
72°C o 7 min. PCR p oduc s we e isualized unde UV ligh a e s aining 5 μl aliquo s wi h
2μl o GelRed™Nucleic Acid Gel S ain (Bio ium, Haywa d, CA, USA) and sepa a ion on a 1%
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 3/14
aga ose gel. Success ully ampli ied p oduc s we e pu i ied using he Exo-SAP p o ocol: he
emaining PCR p oduc (20 μl) was mixed wi h 8 μl o Exo-SAP [5 μl o Exonuclease I
(20 U/μl) (Fe men as,Vilnius, Li huania) and 100 μl o Sh imp Alkaline Phospha ase (1 U/μL)
(Roche Diagnos ics, Indianapolis, USA) in a 1000 μl eac ion mix u e] and incuba ed a 37°C
o 15 minu es and ollowing immedia e incuba ion a 80°C o 15 minu es.
The cycle sequencing eac ions we e pe o med in a 12 μl eac ion mix u e. The eac ion
mix u e con ained 0.5 μl o BigDye
1
Te mina o 3.1 Ready Reac ion mix u e (Pe kin-Elme
Applied Biosys ems, Wa ing on, UK), 2.1 μl o sequencing bu e , 1 μl o ei he he o wa d o
e e se p ime (10 mM) and 2 μl o cleaned PCR p oduc . Sequencing was done in bo h di ec-
ions using same p ime s as used o ampli ica ion. The he mal cycling condi ions o he
sequencing eac ions we e: 25 cycles o 10 s a 96°C, 5 s a 55°C and 4 min a 60°C. The
sequencing p oduc s we e cleaned using e hanol/sal p ecipi a ion and d ied in a lamina low
bench o e nigh . Sequencing was done on an ABI P ism 3100 DNA Analyze (Applied Biosys-
ems, Fos e Ci y, CA, USA) loca ed a he DNA Sequencing Facili y o he Fo es y and Ag i-
cul u al Bio echnology Ins i u e (FABI), Uni e si y o P e o ia.
DNA sequence analyses and ungal iden i ica ion
Geneious R6 so wa e (Bioma e s L d, Auckland, New Zealand) was used o assess he quali y o
sequence ch oma og ams, o edi (when necessa y) and im he 50and 30ends o uni o m leng h,
and o compile he consensus sequences. Fungal iden i ica ion o he species le el was made as a
as possible. This was es ima ed indi idually o each isola e wi h cau ion gi en o he ac ha he
ITS sequence a iabili y in ce ain ungal g oups is low, and acknowledging possibly misiden i ied
sequences in GenBank. Isola es we e iden i ied using a megablas algo i hm implemen ed in Gen-
Bank nucleo ide da abase (h p://blas .ncbi.nlm.nih.go /Blas .cgi). We conside ed eliable iden i-
ica ion o consis o he BLAST ma ches ha had 98% sequence simila i y o ex- ype sequences
o pee - e iewed published s udies. The sequences o isola es ha ep esen ed he same species
based on he BLAST sea ch we e compiled in he same da a se using Molecula E olu iona y
Gene ic Analysis (MEGA) . 6 [32]. The da a se s we e aligned using MAFFT . 7 online e sion
[33] wi h he FFT-NS-i s a egy wi h a 200PAM/κ= 2 sco ing ma ix, a gap opening penal y o
1.53 and an o se alue o 0.00. The sequence simila i ies we e hen isualized and compa ed in
MEGA . 6 and he sequences wi h a 98% simila i y we e assigned o he same species.
S a is ical analyses
The numbe o ungal species pe bee le was analysed using Poisson GLM wi h log link. The
model is speci ied as Y
i
~Poisson(μ
i
), whe e Y
i
is he numbe o ungal species in ba k bee le i,and
mi¼expðb0þb1Siþb2Aiþb2miÞ
whe e S
i
and A
i
a e bina y p edic o s indica ing whe he ba k bee le iwas collec ed in summe
and au umn espec i ely, and m
i
indica es he cen alized numbe o mi es ound in ba k bee le i
(i.e. obse ed numbe o mi es—mean numbe o mi es in he da a); β
0
,β
1
,β
2
,andβ
3
a e he
eg ession coe ficien s associa ed wi h he p edic o s. The same model was fi ed bo h o he o al
numbe o ungal species and o he o al numbe o ophios oma oid species pe ba k bee le.
Poisson GLM is a heo e ically jus i ied model o independen coun s ha do no ha e an
uppe limi [34]. The pa ame e μ
i
speci ies bo h he mean and a iance o he ungal coun o
bee le i. Lack o independence in he da a would lead o o e -dispe sion ( a iance is la ge han
he mean) o unde -dispe sion ( a iance is lowe han he mean), and igno ing exis ing o e -
o unde -dispe sion in modelling leads o p oblems in es s on he eg ession coe icien s. In
his s udy, po en ial o e -dispe sion could be caused by co-associa ion be ween ungal species.
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 4/14
Co espondingly, unde -dispe sion could imply ha some ungi end o exclude each o he .
The model was i ed using he me hod o Maximum Likelihood using R- unc ion glm [35].
Resul s
Collec ion o ba k bee les and mi es
In o al, 298 adul bee les we e collec ed. Du ing he sp ing, 97 bee les we e collec ed, o which
44 we e ound om he soil li e and 53 unde he ba k. Du ing he main swa ming pe iod in
summe , 101 bee les we e collec ed. In he au umn, 100 i s gene a ion bee les we e collec ed,
o which 54 we e om he o es li e and 46 we e ound unde he ba k. Pho e ic mi es we e
ound on 13.1% o adul bee les. The numbe s o mi es ca ied by hese bee les was g ea es in
he au umn, when 22 mi es we e ound, 6 o which we e om he bee les in he li e and 16
we e om he bee les unde he ba k. In he sp ing, 15 mi es we e ound all om bee les in he
li e . In he summe , only 2 mi es we e ound om he dispe sing bee les. When mi es we e
p esen , he numbe pe indi idual bee le anged om 1 o 4, he mean numbe o all bee les
being 0.12 mi es pe bee le.
Isola ion and iden i ica ion o ungi
Fungal associa es we e isola ed om 68.5% o he collec ed bee les and 94.1% o he mi es. The
s udy esul ed in 402 ungal s ains isola ed om he bee les, and 52 s ains isola ed om he
mi es. G ouping o he isola es based on cul u e mo phology esul ed 104 mo phological
g oups. In o al 129 isola es we e selec ed o DNA sequencing. Sequencing he ITS egion
ailed in he case o eigh mo phological g oups. The leng hs o immed consensus sequences
a ied om 500 bp o 650 bp. The sequences ob ained in his s udy we e deposi ed in GenBank
and hei accession numbe s a e p o ided in he Table 1.
Based on he molecula iden i ica ion, ungi isola ed in his s udy we e assigned o 32 ope a-
ional axonomic uni s (OTUs) in ou di e en ungal phyla: Ascomyco a (24 species), Basi-
diomyco a (2 species), Mo ie ellomyco ina (1 species) and Muco omyco ina (5 species)
(Table 1). The ophios oma oid ungi we e he mos nume ous ungi, and ep esen ed as 256
s ains (63.7% o all ungi) isola ed om he bee les, and 45 s ains (86.5% o all ungi) isola ed
om he mi es. Also o he ungi (molds and yeas s) we e equen ly ound bu no eco ded in
his s udy.
Twel e ophios oma oid species we e de ec ed. These included ele en species ha we e
assigned o known species, including Endoconidiopho a polonica,G aphium imb iispo um,
G osmannia cuculla a,G osmannia oli acea,G osmannia penicilla a,G osmannia piceipe da,
Ophios oma ainoae,Ophios oma bicolo ,Ophios oma b unneo-cilia um,Ophios oma piceae,
and Ophios oma e opii (Tables 1and 2). One G osmannia species could no be assigned o
any cu en ly known species, hus ep esen ing a pu a i ely no el axon.
The ITS da a did no p o ide su icien esolu ion o ophios oma oid species delinea ion
wi hin he c yp ic species complexes [36]. The e o e, he ungus iden i ied he e as O.b unneo-
cilia um p obably p esen s a c yp ic no el species simila o O.b unneo-cilia um (Linnakoski
e al. unpublished). O e all, O.bicolo ,O.ainoae,E.polonica and G.piceipe da we e he mos
equen ly isola ed ophios oma oid ungi associa ed wi h he adul bee les (Table 2). These
same species we e he mos equen ly ound isola es associa ed wi h mi es, excep o O.
ainoae ha was ne e de ec ed (Table 3).
The mos nume ous non-ophios oma oid Ascomyco a included Bipola is sp., Bo y is
cine ea and Cladospo ium sp. ha we e p esen easonably equen ly (Table 2). Mos non-
ophios oma oid species we e de ec ed only occasionally. The isola es belonging o
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 5/14

Basidiomyco a, Mo ie ellomyco ina, and Myco omyco ina included species ha we e ypi-
cally p esen only in low numbe s in associa ion wi h adul bee les o hei pho e ic mi es
(Tables 2and 3).
The e we e di e ences be ween he ungal assesmblages om bee les collec ed om di e -
en o e win e ing en i onmen s. Adul s o I. ypog aphus ha o e win e ed in he o es li e
we e exlusi ely o a leas commonly associa ed wi h soil-bo n ungi (e.g. Absidia,Muco and
Umbelopsis)(Table 2). The majo i y o he ophios oma oid species we e de ec ed om bo h
Table 1. Rep esen a i e isola es o I. ypog aphus-associa ed ungi collec ed in his s udy.
Fungal OTU CMW no.
1
GenBank acc. no.
Ascomyco a
Al e na ia a bus i KT896627
A h inium sp. KT896628
Beau e ia pseudobassiana KT896629
Bipola is sp. KT896630
Bo y is cine ea KT896631
Cladospo ium sp. KT896633
Cosmospo a ilio KT896634
Endoconidiopho a polonica 43745 KT896632
G aphium fimb iispo um 43744 KT896635
G osmannia cuculla a 43737 KT896636
G.oli aceae 43741 KT896637
G.penicilla a 43738 KT896638
G.piceipe da 43739 KT896639
G osmannia sp. 43743 KT896640
Monilinia sp. KT896641
Ophios oma ainoae 43718 KT896642
O.bicolo 43723 KT896643
O.b unneo-cilia um KT896644
O.piceae 43732 KT896645
O. e opii 43736 KT896649
Phaeosphae ia agans KT896646
Phaeosphae ia sp. KT896647
Phoma sp. KT896648
T ichode ma polyspo um KT896652
Basidiomyco a
Schizophyllum commune KT896650
T ame es sp. KT896651
Mo ie ellomyco ina
Mo ie ella humilis KT896653
Muco omyco ina
Absidia sp. KT896654
Basidiobolus magnus KT896655
Muco hiemalis .hiemalis KT896656
Umbelopsis isabellina KT896657
Umbelopsis sp. KT896658
1
The cul u e collec ion (CMW) o he Fo es y and Ag icul u al Bio echnology Ins i u e (FABI), Uni e si y o
P e o ia, Sou h A ica
doi:10.1371/jou nal.pone.0155622. 001
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 6/14
Table 2. Seasonal p opo ions o Ips ypog aphus-associa ed ungi isola ed in his s udy.
P opo ion (%)
Fungal OTU Sp ing Summe Au umn To al
li e ba k li e ba k
Ascomyco a
Al e na ia a bus i 0 0 2.13 0 0 1.24
A h inium sp. 0 0 0.85 0 0 0.50
Beau e ia pseudobassiana 0 0 1.70 0 1.41 1.24
Bipola is sp. 1.04 0 13.19 0 1.41 8.21
Bo y is cine ea 1.04 1.04 10.64 0 0 6.72
Cladospo ium sp. 0 0 8.51 0 0 4.98
Cosmospo a ilio 0 0 0 0 1.41 0.25
Endoconidiopho a polonica 3.13 3.13 10.64 4.23 1.41 8.71
G aphium fimb iispo um 2.08 2.08 0.85 1.41 1.41 1.99
G osmannia cuculla a 0 0 0.43 0 1.41 0.50
G.oli aceae 0 0 0.43 0 0 0.25
G.penicilla a 2.08 7.29 0.85 0 1.41 2.99
G.piceipe da 6.25 9.38 2.55 4.23 8.45 7.46
G osmannia sp. 1.04 2.08 2.55 0 1.41 2.49
Monilinia sp. 0 0 2.13 0 0 1.24
Ophios oma ainoae 4.17 4.17 12.77 2.82 0 9.20
O.bicolo 12.50 18.75 22.13 12.68 12.68 24.88
O.b unneo-cilia um 0 0 0.43 0 0 0.25
O.piceae 2.08 2.08 1.70 1.41 0 2.24
O. e opii 1.04 0 0.43 5.63 4.23 2.24
Phaeosphae ia agans 0 0 1.28 0 0 0.75
Phaeosphae ia sp. 0 0 0.43 0 0 0.25
Phoma sp. 1.04 0 0.85 1.41 0 1.00
T ichode ma polyspo um 0 0 0.43 0 0 0.25
Basidiomyco a
Schizophyllum commune 0 1.04 0 0 0 0.25
T ame es sp. 0 0 0.43 0 0 0.25
Mo ie ellomyco ina
Mo ie ella humilis 2.08 1.04 0.85 5.63 2.82 2.74
Muco omyco ina
Absidia sp. 2.08 0 0 8.45 0 1.99
Basidiobolus magnus 0 0 0 1.41 0 0.25
Muco hiemalis .hiemalis 0 0 0.85 4.23 1.41 1.49
Umbelopsis isabellina 6.25 2.08 0 5.63 0 2.99
Umbelopsis sp. 1.04 0 0 0 0 0.25
No bee les 44 53 101 54 46 298
No ungal isola es 47 49 235 41 30 402
No ungal species 16 12 26 13 13 32
Mean no o ophios oma oid species pe bee le 1.50 1.33 1.73 1.38 1.71 1.58
Bee les ca ying a leas one ophios oma oid species (%) 50.00 62.26 76.24 29.63 30.43 54.36
doi:10.1371/jou nal.pone.0155622. 002
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 7/14
o e win e ing subs a es. Amongs he ophios oma oid ungi, E.polonica was ound mo e e-
quen ly om bee les and mi es hibe na ing in he o es li e , and G osmannia species we e
de ec ed mo e commonly om bee les and hei pho e ic mi es hibe na ing unde he ba k
(Tables 2and 3).
Occu ence o ungi in di e en seasons
The ungal species ichness and numbe o isola es de ec ed om I. ypog aphus we e highes
du ing he ba k bee le dispe sal ligh , when in o al 235 isola es assigned o 26 species we e
de ec ed (Table 2). In addi ion, he pe cen age o bee les ca ying ophios oma oid species was
highes in summe , when 76% o he bee les ca ied a leas one ophios oma oid species com-
pa ed o au umn, when only 30% o he bee les we e associa ed wi h ophios oma oid ungi.
Ophios oma bicolo was he p edominan species in all seasons. The o he species mos com-
monly de ec ed included E.polonica,G.piceipe da,G.penicilla a and Umbelopsis isabellina in
he sp ing. Du ing he ba k bee le dispe sal ligh , Bipola is sp., O.ainoae,B.cine ea and E.
polonica we e he mos commonly de ec ed species. Ophios oma e opii and mos species
belonging o Mo ie ellomyco ina and Myco omyco ina we e mainly de ec ed in au umn.
The obse a ions we e suppo ed by he gene alized linea modeling ha iden i ied a clea
associa ion be ween ungal communi ies and season. The mean numbe o ungal species pe
ba k bee le in sp ing was 0.99 species pe bee le. I inc eased o 2.37 (2.4040.985) species in
summe and he ea e dec eased o 0.700 (0.9850.710) species pe bee le in au umn
(Table 4). The di e ence be ween summe and wo o he seasons was highly signi ican
Table 3. Seasonal p opo ions o ungi isola ed om mi es pho e ic on Ips ypog aphus.
P opo ion (%)
Fungal OTU Sp ing Summe Au umn To al
li e ba k li e ba k
Ascomyco a
Cosmospo a ilio 0 0 0 0 2.63 1.92
Endoconidiopho a polonica 16.67 0 0 5.26 0 7.69
G aphium fimb iispo um 8.33 0 0 0 2.63 3.85
G osmannia cuculla a 0 0 0 0 10.53 7.69
G.penicilla a 0 0 0 0 10.53 7.69
G.piceipe da 0 0 50 0 18.42 15.38
G osmannia sp. 0 0 50 0 2.63 3.85
O.bicolo 50.00 0 0 10.53 23.68 36.54
O. e opii 0 0 0 0 10.53 7.69
Mo ie ellomyco ina
Mo ie ella humilis 8.33 0 0 0 0 1.92
Muco omyco ina
Absidia sp. 0 0 0 2.63 0 1.92
Umbelopsis isabellina 16.67 0 0 0 0 3.85
No mi es 15 0 2 6 16 39
No ungal isola es 12 0 2 7 31 52
No ungal species 502 3812
Mean no o ophios oma oid species pe mi e 0.9 0 2.0 1.67 1.5 1.40
Mi es ca ying a leas one ophios oma oid species (%) 75 0 50 83.33 93.75 76.92
doi:10.1371/jou nal.pone.0155622. 003
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 8/14
(p<0.000) and he di e ence be ween sp ing and au umn was signi ican (p = 0.029). The
numbe o mi es did no ha e s a is ically signi ican (p = 0.259) posi i e e ec on he numbe
o ungal species pe bee le. Ve y simila esul s we e ob ained o ophios oma oid ungi spe-
cies, whe e he mean numbe o ungal species in sp ing, summe and au umn we e 0.79, 1.33
and 0.45, espec i ely, and he di e ences be ween seasons we e highly signi ican . The da a
did no show clea signs o o e - o unde -dispe sion, he e o e he da a did no indica e co-
exis ence o exclusion o ungal species.
Discussion
Clea associa ions we e ound be ween ungal communi ies and season in his s udy and hese
coincide wi h he li e s ages o I. ypog aphus. This sugges s seasonal succession among I. ypo-
g aphus associa ed ungi. The da a showed ha I. ypog aphus ec o s a la ge di e si y o ungi.
O hese, a o al o 32 ope a ional axonomic uni s (OTUs) esided in ou ungal phyla/sub-
phyla (24 Ascomyco a, 2 Basidiomyco a, 5 Muco omyco ina and 1 Mo ie ellomyco ina) based
on he ungal ba code (ITS) gene egion. The s udy is also he i s o p o ide p elimina y
in o ma ion ega ding he ungi associa ed wi h mi es pho e ic on I. ypog aphus in Finland.
The ungal species ichness and he numbe o ungal isola es a ied be ween he seasons
sampled. Some o he ungal species we e de ec ed only in a ce ain season, e.g. Schizophyllum
commune in he sp ing, Al e na ia a bus i in he summe , and Cosmospo a ilio in he
au umn. The majo i y o he ophios oma oid ungi we e p esen in all he seasons. Some o
hese ungi we e de ec ed mo e commonly in a speci ic season, e.g. G.penicilla a in he sp ing,
E.conidiopho a in he summe , and O. e opii in he au umn. The bee le’s o e win e ing si e
also a ec ed he ungal associa es o I. ypog aphus. In e es ingly, G osmannia species we e
ound mo e commonly on bee les and mi es ha hibe na ed unde he hos ee’s ba k. This
obse a ion suppo s he esul s o a p e ious s udy whe e G osmannia species we e mo e
o en ound on bee les collec ed unde he ba k [21]. Mos o he ungi we e de ec ed du ing
he dispe sal ligh pe iod o I. ypog aphus. The high numbe o di e en ungi du ing he dis-
pe sal ligh is mos p obably due he ac ha I. ypog aphus can dispe se o e long dis ances
[37–40]. The e o e, he indi iduals caugh in his s udy would likely ha e o igina ed om qui e
a la ge a ea. In e es ingly, he indi idual ungal species occu ed independen ly o each o he .
This is consis en wi h he esul s o a p e ious s udy whe e no co ela ion was ound among
di e en ungal species associa ed wi h I. ypog aphus [41].
The ungal species assemblage associa ed wi h I. ypog aphus in he p esen s udy was con-
sis en wi h ha ound in p e ious s udies conduc ed in Eu ope [11–12,15,19–22,26–27,41–
49]. The ungal assemblage is known o consis mainly o ascomyce es, which we e also he
mos common ungi in his s udy. O hese, he biodi e si y o ophios oma oid ungi belonging
o o de s Mic oascales and Ophios oma ales (Ascomyco a) a e bes known in his niche [10,
50] and hey we e he mos nume ous species ound in his s udy. The ungi de ec ed in low
Table 4. Pa ame e es ima es (displayed agains sp ing) o he Poisson GLM o all ungi (le ) and o ophios oma oid ungi.
All ungi Ophios oma oid ungi
^
βexp ð^
βÞs.e. p- alue ^
βexp ð^
βÞs.e. p- alue
Cons an -0.015 0.985 0.102 0.883 -0.239 0.788 0.115 0.037
Summe 0.877 2.404 0.123 0.000 0.524 1.688 0.145 0.000
Au umn -0.342 0.710 0.157 0.029 -0.559 0.572 0.187 0.003
Mi es 0.120 1.128 0.106 0.259 0.157 1.170 0.118 0.184
doi:10.1371/jou nal.pone.0155622. 004
Seasonal Succession among Ips ypog aphus-Associa ed Fungi
PLOS ONE | DOI:10.1371/jou nal.pone.0155622 May 17, 2016 9/14