A Rela ionship be ween Ca o enoid Accumula ion and
he Dis ibu ion o Species o he Fungus
Neu ospo a
in
Spain
E a M. Luque, Gab iel Gu ie
´ ez, Lau a Na a o-Samped o
¤a
, Ma ı
´a Olmedo
¤b
, Julio Rod ı
´guez-
Rome o
¤c
, Ca men Ruge -He e os, Vı
´c o G. Tagua, Luis M. Co ochano*
Depa amen o de Gene
´ ica, Uni e sidad de Se illa, Se illa, Spain
Abs ac
The ascomyce e ungus Neu ospo a is p esen in many pa s o he wo ld, in pa icula in opical and sub opical a eas,
whe e i is ound g owing on ecen ly bu ned ege a ion. We ha e sampled he Neu ospo a popula ion ac oss Spain. The
sampling si es we e loca ed in he egion o Galicia (no hwes e n co ne o he Ibe ian peninsula), he p o ince o Ca
´ce es,
he ci y o Se ille, and he wo majo islands o he Cana y Islands a chipelago (Tene i e and G an Cana ia, wes coas o
A ica). The si es co e ed a la i ude in e al be ween 27.88uand 42.74u. We ha e iden i ied wild- ype s ains o N. disc e a,N.
e aspe ma,N. c assa, and N. si ophila and he equency o each species a ied om si e o si e. I has been shown ha
a e exposu e o ligh Neu ospo a accumula es he o ange ca o enoid neu ospo axan hin, p esumably o p o ec ion om
UV adia ion. We ha e ound ha each Neu ospo a species accumula es a di e en amoun o ca o enoids a e exposu e o
ligh , bu hese di e ences did no co ela e wi h he exp ession o he ca o enogenic genes al-1 o al-2. The accumula ion
o ca o enoids in Neu ospo a shows a co ela ion wi h la i ude, as Neu ospo a s ains isola ed om lowe la i udes
accumula e mo e ca o enoids han s ains isola ed om highe la i udes. Since egions o low la i ude ecei e high UV
i adia ion we p opose ha he inc eased ca o enoid accumula ion may p o ec Neu ospo a om high UV exposu e. In
suppo o his hypo hesis, we ha e ound ha N. c assa, he species ha accumula es mo e ca o enoids, is mo e esis an
o UV adia ion han N. disc e a o N. e aspe ma. The pho op o ec ion p o ided by ca o enoids and he capabili y o
accumula e di e en amoun s o ca o enoids may be esponsible, a leas in pa , o he dis ibu ion o Neu ospo a species
ha we ha e obse ed ac oss a ange o la i udes.
Ci a ion: Luque EM, Gu ie
´ ez G, Na a o-Samped o L, Olmedo M, Rod ı
´guez-Rome o J, e al. (2012) A Rela ionship be ween Ca o enoid Accumula ion and he
Dis ibu ion o Species o he Fungus Neu ospo a in Spain. PLoS ONE 7(3): e33658. doi:10.1371/jou nal.pone.0033658
Edi o : Sco E. Bake , Paci ic No hwes Na ional Labo a o y, Uni ed S a es o Ame ica
Recei ed July 28, 2011; Accep ed Feb ua y 17, 2012; Published Ma ch 20, 2012
Copy igh : ß2012 Luque 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 wo k was suppo ed by he Spanish ‘‘Minis e io de Ciencia e Inno acio
´n’’ (INIA RM2004-0007, BIO2009-12486), and Jun a de Andalucı
´a (P09-CVI-
5027). These g an s a e suppo ed by he Eu opean Regional De elopmen Fund. CRH is a esea ch ellow o he Regional Go e nmen (Jun a de Andalucı
´a). 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: co [email protected]
¤a Cu en add ess: Cen o de In es igacio
´n Tecnologı
´a e Inno acio
´n de la Uni e sidad de Se illa (CITIUS), Uni e sidad de Se illa, Se illa, Spain
¤b Cu en add ess: Depa men o Molecula Ch onobiology, Uni e si y o G oningen, G oningen, The Ne he lands
¤c Cu en add ess: Depa men o Mic obiology, Ka ls uhe Ins i u e o Technology (KIT), Ka ls uhe, Ge many
In oduc ion
The ascomyce e ungus Neu ospo a c assa is used as a model
o ganism o esea ch on di e en aspec s o euka yo ic molecula
biology including RNA inac i a ion and gene silencing, he
mechanism o gene ic ecombina ion, egula ion by he ci cadian
clock, and he egula ion by ligh o gene exp ession [1–4]. In
addi ion, a collec ion o mo e han 4000 Neu ospo a wild- ype
s ains om na u al popula ions has p o ided in o ma ion abou
he dis ibu ion o he di e en species o Neu ospo a in he wo ld
[5,6]. Genomic analysis o wild- ype s ains o Neu ospo a has
allowed de ailed cha ac e iza ion o he p ocess o adap a ion o
local en i onmen al condi ions [7]. Mos o he Neu ospo a species
ha e been iden i ied in opical o sub opical a eas whe e hey
can be easily spo ed g owing on he su ace o ecen ly bu ned
ege a ion. Ex ensi e su eys ha e ex ended he geog aphical
dis ibu ion o Neu ospo a o empe a e a eas o wes e n No h
Ame ica and Eu ope, wi h colonies o N. disc e a iden i ied as a
no h as Alaska [8,9]. The dis ibu ion o Neu ospo a species in
na u e is a ied. Fo example, N. disc e a is he mos equen
Neu ospo a species isola ed in wes e n No h Ame ica [8] bu in
Eu ope N. disc e a was a ely ound while N. c assa,N. si ophila, and
N. e aspe ma we e equen ly obse ed [9].
The colonies o Neu ospo a a e e y conspicuous due o he
accumula ion o he o ange ca o enoid neu ospo axan hin in
conidia and ege a i e mycelia [10]. The biosyn hesis o
neu ospo axan hin in ege a i e mycelia is induced by ligh [11]
h ough he ac i a ion o he biosyn he ic genes [12,13]. Ligh
se es as an en i onmen al cue o adjus he ci cadian clock so ha
Neu ospo a can an icipa e changes in en i onmen al condi ions
[12,14–16]. Ca o enoids, like neu ospo axan hin, a e an ioxidan s
due o hei capaci y o quench eac i e oxygen species [17–19],
and p o ide p o ec ion agains UV damage in human skin [20]
and ungi [21–23], bu no agains gamma- adia ion in he ungus
Phycomyces blakesleeanus [24]. Pigmen ed s ains o he basidiomy-
ce ous yeas Spo obolomyces ube imus and Cys o ilobasidium capi a um
PLoS ONE | www.plosone.o g 1 Ma ch 2012 | Volume 7 | Issue 3 | e33658
we e mo e ole an o UV damage and showed be e su i al
a e UV ea men han unpigmen ed s ains [21]. The
accumula ion o ca o enoids p o ec ed he yeas Rhodo o ula
mucilaginosa om UV damage [23]. In animals, a pho op o ec i e
ole o ca o enes in echinoids eggs has been sugges ed [25], and
mice ed wi h be a-ca o ene o can haxan hin we e p o ec ed
agains skin umo s caused by UV adia ion [26]. Ca o enoids
p o ide p o ec ion agains excess i adia ion in pho osyn he ic
o ganisms. Fo example, accumula ion o ca o enoids (be a-
ca o ene, zeaxan hin, o can haxan hin) in s ains o he
cyanobac e ium Synechococcus a e ans o ma ion wi h ca o eno-
genic genes led o p o ec ion o pho osyn hesis om UV damage
[27,28]. The ac i a ion by ligh o ca o enoid biosyn hesis in
Neu ospo a and o he ungi [29] may op imize p o ec ion agains
UV adia ion when he ungus is g owing exposed o ligh in open
en i onmen s. T opical egions (low la i ude) ecei e mo e sola
adia ion han no he n loca ions (high la i ude). Fo a gi en si e
he amoun o sola adia ion changes du ing he ime o he yea ,
he al i ude and he a mosphe ic condi ions, bu du ing he
summe low-la i ude loca ions ecei e wice he amoun o UV-B
adia ion (280–315 nm) han high-la i ude loca ions [30,31]. I is
possible ha he capabili y o accumula e ca o enoids may a ec
he dis ibu ion o Neu ospo a species in low-la i ude a eas due o a
high exposu e o UV adia ion.
The egion o Galicia (no hwes e n co ne o he Ibe ian
peninsula) and he wo majo islands o he Cana y Islands
a chipelago (Tene i e and G an Cana ia, wes coas o A ica)
su e ed an unusual numbe o wild i es du ing he summe s o
2006 and 2007 espec i ely. Since Neu ospo a is easily spo ed on
bu ned ege a ion, hese summe i es allowed he oppo uni y o
sample he Neu ospo a popula ions in hese sepa a ed a eas. We
ha e obse ed di e ences in he dis ibu ion o Neu ospo a species in
each si e. The ou Neu ospo a species ha we ha e iden i ied (N.
disc e a,N. c assa,N. e aspe ma, and N. si ophila) showed di e ences
in he accumula ion o ca o enoids a e ligh exposu e. In
addi ion, we ha e ound a co ela ion be ween he accumula ion
o ca o enoids in Neu ospo a and he la i ude o he sampling si e.
Ou esul s sugges ha he capabili y o accumula e ca o enoids
plays a ole in he dis ibu ion o Neu ospo a species in na u e.
Resul s and Discussion
Neu ospo a in Spain
Colonies o Neu ospo a we e eadily obse ed g owing on he
su ace o pa ially bu ned ege a ion (Fig. 1), as al eady obse ed
p e iously in o he Eu opean collec ion si es [9]. We collec ed
Neu ospo a wild- ype s ains om eigh si es loca ed in he egion o
Galicia and om one si e in he p o ince o Ca´ce es (summe o
2006), and om six si es in he Cana y Island a chipelago (summe
o 2007) ha included samples om he wo majo islands
(Tene i e and G an Cana ia). The si es we e selec ed by hei
accessibili y o loca ions whe e wild i es had occu ed ecen ly (4–
6 weeks). The si es ha we selec ed may no be ep esen a i es o
he en i e egion. The loca ion o he collec ion si es had la i udes
ha anged om 27.88uN (Fa aga and Mo a´n, Las Palmas) o
42.74uN (He bo´n, A Co un˜a). In 41 plan s we ook mo e han one
sample in o de o in es iga e he p esence o gene ic a ia ions in
he Neu ospo a s ains ha colonized a single plan . The samples
we e collec ed, pu i ied om single colonies, and s o ed p io o
u he cha ac e iza ion. F om he samples ha we isola ed in he
ield ips we pu i ied and s o ed a o al o 125 wild- ype s ains
(Table S1). Ou collec ion also includes 26 Neu ospo a c assa s ains
isola ed om Se ille in 2004 ha ha e been epo ed p e iously
[9].
Iden i ica ion o Neu ospo a species
We used a phylogene ic me hod o iden i y he species o each
collec ed Neu ospo a s ain. We ampli ied and sequenced h ee
unlinked polymo phic loci (TMI, TML, and DMG) om each
wild- ype s ain [32]. These loci ha e been used success ully o
in e he e olu ion and di e si y o species o he genus Neu ospo a
[32–35]. The sequences om he h ee loci om each s ain we e
combined and aligned wi h homologous sequences om a se o
e e ence Neu ospo a s ains [32,35]. The esul ing DNA alignmen
was used o econs uc a phylogene ic ee by he Maximum-
Likelihood and Neighbo -Joining me hods (Fig. 2). The Neu ospo a
species o each isola e was deduced by he p esence o a e e ence
Neu ospo a species close o each unknown s ain in he phylogene ic
ee. A g oup o isola es o med a clade wi h di e en N. c assa
e e ence s ains and we e assigned o N. c assa. The Spanish N.
c assa o med a clade wi h a e e ence N. c assa s ain om clade B
as p e ious Eu opean N. c assa isola es [9]. Simila ly, a g oup o
isola es o med a clade wi h e e ence N. disc e a s ains and we e
assigned as N. disc e a. Finally, one s ain (GC4-5C) o med a clade
wi h N. si ophila and was assigned as a N. si ophila (Fig. 2).
Some s ains could no be assigned using he phylogene ic
me hod. Th ee isola es (C9C, OU12C, and C6A) o med a well-
de ined clade ha sepa a ed be o e he majo clade ha included
all he N. disc e a e e ence s ains (Fig. 2). These s ains we e
classi ied as N. disc e a bu hey may ep esen a new phylogene ic
Figu e 1.
Neu ospo a
g owing on he unk o a bu ned ee.
Colonies o conidia ing Neu ospo a a e easily spo ed by hei o ange
colo due o he accumula ion o ca o enoid pigmen s. The pic u e was
aken in a ga den in Se ille (Spain) a ew weeks a e a summe i e in
2004 and is p obably Neu ospo a c assa as all he samples aken om
his si e we e la e iden i ied as belonging o his species.
doi:10.1371/jou nal.pone.0033658.g001
The Fungus Neu ospo a in Spain
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species. In addi ion, a g oup o s ains o med a clade ha
included N. e aspe ma, and N. hispaniola (Fig. 2). As an al e na i e
me hod o iden i y he Neu ospo a species co esponding o hese
s ains we pe o med blas sea ches using DNA sequences o he
TMI, TML, and DMG loci om each s ain. Sea ches o he
Neu ospo a DNA da abase wi h sequences om DMG and TML
om s ains C9C, OU12C, and C6A iden i ied N. disc e a DNAs as
he mos simila sequences, hus con i ming hese s ains as N.
disc e a. Sea ches using TMI iden i ied a a ie y o di e en
Neu ospo a species and we e no conside ed. Sea ches using he
h ee loci om he g oup o pu a i e N. e aspe ma s ains we e no
conclusi e as hey iden i ied a a ie y o di e en Neu ospo a
species. We he e o e ampli ied and sequenced a ou h locus
(QMA) [32] om hese s ains. Blas sea ches using QMA
Figu e 2. Iden i ica ion o
Neu ospo a
species by phylogeny. Maximum-Likelihood ee p oduced om he TMI, DMG and TML loci combined.
B anch suppo alues (Maximum-likelihood boo s ap p opo ions/Neighbo -Joining boo s ap p opo ions) in combined analyses a e displayed o
majo b anches only. The well-suppo ed g oups o indi iduals a e indica ed by iangles, wi h heigh p opo ional o numbe o indi iduals and
wid h p opo ional o he mean numbe o changes om he node. Only boo s ap p opo ions g ea e han 50% a e shown. The numbe o isola es
in shown in pa en hesis.
doi:10.1371/jou nal.pone.0033658.g002
The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 3 Ma ch 2012 | Volume 7 | Issue 3 | e33658
sequences om hese s ains iden i ied N. e aspe ma DNA in he
Neu ospo a DNA da abase and suppo ed he assignmen o hese
s ains as N. e aspe ma. In addi ion, hese s ains p oduced
Neu ospo a sexual s uc u es, pe i hecia, when g own in indepen-
den cul u es. Ma u ed pe i hecia con ained asci wi h ou
ascospo es, and he s ains de eloped asexual conidia (no shown)
and accumula ed ca o enoids (see below). These biological
p ope ies a e speci ic o N. e aspe ma, a sel e ile species ha
can ep oduce in isola ion due o he p esence o nuclei wi h ei he
ma ing ype in he same hyphae (pseudohomo halism) [5]. The
biological p ope ies o hese s ains, and he simila i ies o hei
QMA locus wi h N. e aspe ma DNA suppo ed ou assignmen o
hese s ains as N. e aspe ma. Fu he cha ac e iza ion o he
Neu ospo a wild- ype s ains by phylogene ic species ecogni ion
(PSR) [36] con i med he Neu ospo a species assigned o each s ain,
wi h he excep ion o he N. e aspe ma s ains.
Dis ibu ion o Neu ospo a species in Spain
We ha e iden i ied 64 N. disc e a s ains, 17 N. e aspe ma s ains,
69 N. c assa s ains, and one N. si ophila s ain (Table 1, Table S1).
These he e o hallic and pseudohomo hallic species appea as a
e minal clade in he phylogeny o he genus Neu ospo a [37]. The
dis ibu ion o Neu ospo a species ac oss he si es ha we su eyed
was a ied, pe haps e lec ing di e ences in humidi y, al i ude,
and ligh exposu e (Fig. 3). N. disc e a was only ound in Galicia and
Ca´ce es, bu was absen om sou he n si es. On he con a y, N.
c assa was ound in Se ille and he Cana y Island si es, bu no in
he no he n si es ha we sampled. N. e aspe ma showed a wide
dis ibu ion as i was iden i ied in Galicia and he Cana y Islands
(Fig. 3).
In 10 ou o he 16 si es su eyed we ound a single Neu ospo a
species, he o he six si es had ei he N. disc e a o N. c assa and N.
e aspe ma (N. si ophila was isola ed in Fa aga, a si e whe e we also
ound N. c assa and N. e aspe ma). P e ious cha ac e iza ion o
Neu ospo a in Eu ope allowed he iden i ica ion o a single species in
only ou ou o 14 si es, he e o e mos si es had mul iple
Neu ospo a species [9]. Ou disco e y o a single isola e o N. si ophila
in he Cana y island o G an Cana ia was unusual, as se e al N.
si ophila s ains ha e been isola ed in Spain and Po ugal, and in
o he pa s o Eu ope. N. si ophila ep esen ed 34% o all he
Neu ospo a isola es in a Eu opean su ey [9], and i s nea ly absence
in ou su ey sugges s ha N. si ophila may ha e a es ic ed habi a
ha was no included in ou ield ips.
Mos o he 41 plan s whe e we isola ed mul iple independen
samples we e colonized by a single Neu ospo a species. In ou cases
we isola ed wo di e en Neu ospo a species g owing in he same
plan ( h ee plan s had N. c assa and N. e aspe ma, and one plan
had N. c assa and N. si ophila). In six addi ional cases we ound
plan s colonized by gene ically di e en indi iduals (haplo ypes) o
N. disc e a, esul ing in a o al o 10 ou o 41 plan s colonized wi h
mo e han one Neu ospo a indi idual. This alue is likely an
unde es ima e o he equency o plan s colonized by di e en
Neu ospo a indi iduals as we sequenced a e y mino pa o he
genome, and we a bi a ily de ined gene ically di e en indi id-
uals as hose ha had mo e han 5% di e ences in he sequence o
hei h ee polymo phic loci (TMI, TML, and DMG). Ou esul s
show ha mo e han one Neu ospo a indi idual can colonize and
comple e a ege a i e cycle in a small a ea (a single plan )
suppo ing p e ious obse a ions [9,38].
Accumula ion o ca o enoids in Neu ospo a species
Du ing he p ocess o isola ion and cha ac e iza ion o he
Neu ospo a wild- ype s ains we no iced di e ences in he amoun o
ca o enoids accumula ed. We hen assayed he amoun o
ca o enoids accumula ed by mycelia om each Neu ospo a s ain
a e exposu e o ligh du ing one day as compa ed o he
accumula ion obse ed in mycelia kep in he da k. We obse ed
ha all he s ains had aces o ca o enoids in mycelia kep in he
da k (3–6 mg/g d y mass) bu hey di e ed in he amoun o
ca o enoids accumula ed a e ligh exposu e, and his di e ence
Table 1. Dis ibu ion o species o Neu ospo a ac oss si es in Spain.
P o ince Si e La i ude Longi ude Al i ude
a
N. disc e a N. e aspe ma N. c assa N. si ophila
To al
A Co un
˜aHe bo
´n 42.74u28.62u79 16 16
Pon e ed a Co obad 42.46u28.46u444 1 1
Lagoas 42.46u28.50u344 3 1 4
O G o e 42.42u28.84u94 3 1 4
Ou ense Rouzo
´s 42.42u27.92u457 9 9
Lin
˜a es 42.40u27.92u407 6 2 8
A Gudin
˜a 42.05u27.12u1075 11 11
Lamas 41.98u27.54u709 7 7
Ca
´ce es Can
˜a e al 39.80u26.38u381 9 9
Se illa
b
Se illa 37.37u25.99u82626
S. C. de Tene i e La Guancha 28.37u216.65u504 1 10 11
Los Realejos 28.36u216.58u737 3 3
Masca 28.30u216.83u1029 1 3 4
S. del Teide 28.29u216.81u908 2 2
Las Palmas Moga
´n 27.88u215.72u332 3 3
Fa aga 27.88u215.56u638 10 22 1 33
To al 64 17 69 1 151
a
Me e s.
b
The iden i ica ion o hese s ains has been epo ed p e iously [9].
doi:10.1371/jou nal.pone.0033658. 001
The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 4 Ma ch 2012 | Volume 7 | Issue 3 | e33658
was speci ic o each Neu ospo a species (Table 2, Table S1). N.
disc e a was he species ha accumula ed less ca o enoids a e ligh
exposu e (22.7 mg/g d y mass), while N. e aspe ma (100.9 mg/g d y
mass) and N. c assa (141.1 mg/g d y mass) showed highe
accumula ions o ca o enoids. The only N. si ophila s ain ha we
isola ed accumula ed 142.7 mg/g d y mass o ca o enoids. Fo a
compa ison he N. c assa s anda d wild- ype s ain (74-OR23-1VA)
accumula ed 225.2 mg/g d y mass o ca o enoids (a e age o wo
expe imen s). Ou esul s show ha he Neu ospo a species ha we
ha e es ed di e ed in hei capabili ies o accumula e ca o enoids
a e ligh exposu e.
The di e ences in he accumula ion o ca o enoids by he
species o Neu ospo a did no co ela e wi h he exp ession o genes
al-1 o al-2 (Fig. 4). These genes encode enzymes equi ed o he
biosyn hesis o neu ospo axan hin. Gene al-1 encodes he
phy oene dehyd ogenase [39], and al-2 is a bi unc ional gene
esponsible o he phy oene syn hase and he ca o ene cyclase
[40,41]. The exp ession o hese wo genes is induced by ligh and
we obse ed simila le els o ligh -dependen mRNA accumula-
ion in s ains ha accumula e low o high amoun s o ca o enoids
a e exposu e o ligh (Fig. 4). Fo example, he ligh -dependen
al-2 mRNA accumula ion in s ain C11A (N. disc e a) ha
accumula ed 12.5 mg/g d y mass o ca o enoids was simila o
he ela i e al-2 mRNA amoun obse ed in s ain TF2-6A (N.
c assa) ha accumula ed 240.2 mg/g d y mass o ca o enoids.
Simila ly, we did no obse e changes in he ligh -dependen al-1
mRNA accumula ion be ween s ains C11A and PO9C (N. disc e a)
despi e he en- old di e ence in ca o enoid accumula ion (Fig. 4).
These esul s indica e ha changes in gene exp ession, a leas o
al-1 and al-2, a e no esponsible o he di e ences in he inal
amoun o ca o enoids accumula ed a e exposu e o ligh ha we
ha e obse ed in di e en species o Neu ospo a. I is possible ha
al e na i e mechanisms, he ac i a ion by ligh o o he key genes
o he ligh -dependen modi ica ion o egula o y p o eins, play a
majo ole in he egula ion o ca o enoid accumula ion in
Neu ospo a.
The accumula ion o ca o enoids in Neu ospo a shows a
co ela ion wi h la i ude
The amoun o ca o enoids ha N. disc e a s ains accumula e
a e ligh exposu e a ied li le ega dless o hei isola ion si e.
Howe e , we no iced ha N. e aspe ma and N. c assa s ains
isola ed in di e en loca ions di e ed in he amoun o ca o enoids
accumula ed a e exposu e o ligh (Table 2, Table S1). Fo
example, he N. e aspe ma s ains isola ed in Galicia accumula ed
66.7 mg/g d y mass in a e age compa ed o he 115.2 mg/g d y
mass accumula ed by he N. e aspe ma s ains isola ed in he
Cana y Islands (Table 2). Simila ly, he N. c assa s ains isola ed in
Se ille accumula ed 108.6 mg/g d y mass in a e age while he N.
c assa s ains isola ed in he Cana y Islands accumula ed 160.7 mg/
g d y mass in a e age (Table 2). These esul s show ha he
Neu ospo a s ains isola ed om lowe la i udes ( he Cana y Islands)
had he capabili y o accumula e mo e ca o enoids a e exposu e
o ligh han he Neu ospo a s ains isola ed om highe la i udes
(Galicia and Se ille).
In o de o explo e in mo e de ail he ela ionship be ween
ca o enoid accumula ion and he loca ion o he isola ion si e we
plo ed he amoun o ca o enoids accumula ed a e exposu e o
ligh and he la i ude o he collec ion si e o he 150 wild- ype
s ains ha we ha e isola ed (Fig. 5). The plo shows a nega i e
co ela ion be ween ca o enoid accumula ion and la i ude (coe -
Table 2. Accumula ion o ca o enoids in species o Neu ospo a isola ed om Spain.
Ca o enoids
a
Numbe o s ains
La i ude Ligh Da k
N. disc e a 42.74u-39.80u22.761.5 3.960.2 63
N. e aspe ma 42.46u-27.88u100.968.4 4.960.5 17
N. c assa 37.37u-27.88u141.165.2 5.460.2 69
N. si ophila 27.88u142.760.0 6.760.0 1
N. disc e a Galicia 42.74u-41.98u23.461.7 4.160.2 54
N. disc e a Ca
´ce es 39.80u18.161.0 3.060.2 9
N. e aspe ma Galicia 42.46u-42.40u66.763.8 3.160.3 5
N. e aspe ma Cana y Islands 28.37u-27.88u115.268.9 5.660.6 12
N. c assa Se ille 37.37u108.665.5 6.060.5 26
N. c assa Cana y Islands 28.37u-27.88u160.765.9 5.060.2 43
a
A e age6SEM (mg/g d y mass).
doi:10.1371/jou nal.pone.0033658. 002
Figu e 3. Dis ibu ion o
Neu ospo a
species collec ed in Spain.
doi:10.1371/jou nal.pone.0033658.g003
The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 5 Ma ch 2012 | Volume 7 | Issue 3 | e33658

icien o de e mina ion R
2
= 0.71) and con i ms he obse a ion
ha Neu ospo a s ains isola ed om lowe la i udes accumula e
mo e ca o enoids a e exposu e o ligh han s ains isola ed om
highe la i udes. A wo-way analysis o a iance (ANOVA)
be ween Neu ospo a species, la i ude, and ca o enoid accumula ion
con i med ha Neu ospo a species showed signi ican di e ences in
ca o enoid accumula ion, and ha he species isola ed om
di e en la i udes showed signi ican di e ences in ca o enoid
accumula ion (p,0.001) (Table S2).
The nega i e co ela ion shown in Fig. 5 can be explained by
he combina ion o wo e ec s. Fi s , we only de ec ed N. disc e a
s ains in high la i ude si es, and hese s ains accumula ed less
ca o enoids han N. e aspe ma and N. c assa (Table 2). N. c assa, he
species wi h he highes accumula ion o ca o enoids, was only
de ec ed in low-la i ude si es. Second, N. e aspe ma and N. c assa
s ains isola ed in low-la i ude si es accumula ed mo e ca o enoids
a e exposu e o ligh han s ains o he same species isola ed
om high-la i ude si es (Table 2).
The si es ha we ha e su eyed di e ed in many cha ac e is ics
in addi ion o la i ude, including al i ude, a e age empe a u e,
and humidi y. We did no de ec any ela ionship be ween al i ude
and ca o enoid accumula ion (no shown), bu o he en i onmen-
al ac o s and di e ences in ege a ion may ha e con ibu ed o
he co ela ion ha we ha e unco e ed.
The e is a co ela ion be ween sola adia ion and la i ude as
a eas o low la i ude ecei e mo e UV adia ion han a eas o high
la i ude [30,31]. UV adia ion damages DNA by inducing he
o ma ion o cyclobu ane-py imidine dime s and 6-4 pho op od-
uc s [42,43], and cells ha e molecula mechanisms o p o ec
DNA om UV-induced damage [43]. In ungi he ha m ul e ec s
o UV adia ion a e educed a e he ac i a ion by ligh o he
biosyn hesis o sc eening and pho op o ec i e pigmen s, like
ca o enoids, and he ac i a ion by ligh o genes o DNA epai
[11,29,44–47]. As UV i adia ion is highe in egions o low
la i ude han in high-la i ude loca ions we p opose ha he
inc eased ca o enoid accumula ion ha we ha e obse ed in
Neu ospo a s ains isola ed om lowe la i udes may p o ide an
inc eased p o ec ion om high UV exposu e. The pho op o ec-
ion p o ided by ca o enoids and he capabili y o accumula e
di e en amoun s o ca o enoids may be, a leas in pa ,
esponsible o he dis ibu ion o Neu ospo a species ha we ha e
obse ed in se e al a eas o Spain. An al e na i e hypo hesis
would be ha o he unknown ac o s played majo oles in he
dis ibu ion o Neu ospo a species in na u e and ha he species
inc eased hei ca o enoid accumula ion du ing he p ocess o
adap a ion o low la i udes while ca o enoid accumula ion
emained less ele an in species adap ed o high-la i ude
loca ions.
Conidia o N. c assa a e mo e esis an o UV adia ion
han conidia om N. disc e a o N. e aspe ma
In o de o explo e in mo e de ail he ole o ca o enoids in UV
p o ec ion we assayed he su i al o conidia om se e al
Neu ospo a species a e UV exposu e (Fig. 6). To pe o m he
assay we selec ed s ains ha accumula ed low o high amoun o
ca o enoids and he s anda d wild- ype s ain o a compa ison.
The capabili y o accumula e ca o enoids in mycelia a e
exposu e o ligh did no p o ide addi ional p o ec ion o UV
exposu e in ou assay. Fo example s ain GC4-1A (N. c assa)
showed be e su i al a e UV exposu e han s ain GC4-4C (N.
e aspe ma), bu GC4-1A accumula ed less ca o enoids han GC4-
4C a e exposu e o ligh (Fig. 6). I should be no ed ha his assay
was pe o med wi h conidia, and Neu ospo a conidia accumula e
ca o enoids cons i u i ely due o he exp ession o he al genes
[48,49]. I is possible ha he capabili y o accumula e ca o enoids
a e exposu e o ligh is mo e impo an o he su i al o
ege a i e mycelia a e UV i adia ion han o he su i al o
conidia. The h ee wild- ype s ains o N. c assa ha we assayed
showed be e su i al a e UV exposu e han s ains o N.
e aspe ma o N. disc e a, in pa icula a e ou min o UV exposu e
(Fig. 6). This could be due o he p esence o high amoun s o
ca o enoids in conidia, bu o he al e na i es like an imp o ed
machine y o DNA epai canno be uled ou . The g ea e
esis ance o N. c assa conidia o UV i adia ion, he p edominance
o his species a collec ion si es a lowe la i udes, and hei abili y
o p oduce he mos ca o enoids, suppo he hypo hesis ha he
dis ibu ion o Neu ospo a species ac oss la i udes is in luenced by
ligh -induced ca o enoid p oduc ion.
A ela ionship be ween esis ance o UV exposu e and he
la i ude o he isola ion si e has been shown o he en omopa ho-
genic ungi Beau e ia bassiana and Me a hizium anisopliae as s ains
isola ed close o he equa o showed imp o ed esis ance o UV
han s ains isola ed a highe la i udes [50,51]. These obse a ions
sugges ha esis ance o UV adia ion play a ole in he
dis ibu ion o ungi in na u e, a leas o he small numbe o
examples in es iga ed.
N. c assa,N. disc e a, and N. e aspe ma ha e been isola ed in
addi ional loca ions ac oss Eu ope [9], and N. disc e a has been
Figu e 4. Ac i a ion by ligh o he
albino
genes in
Neu ospo a
species. Quan i a i e RT-PCR expe imen s we e pe o med o measu e
he ela i e accumula ion o al-1 o al-2 mRNA in mycelia o wild- ype
s ains exposed o whi e ligh (2 W/m
2
blue ligh ) o kep in he da k.
The plo s show he a e age and s anda d e o o he mean o he
ela i e mRNA accumula ion in ou independen expe imen s, each
wi h h ee eplica es. The esul s om each PCR o each gene we e
no malized o he co esponding PCR o ub-2 o co ec o sampling
e o s and no malized o he esul ob ained wi h mycelia kep in he
da k. The amoun o ca o enoids accumula ed by each wild- ype s ain
in cul u es exposed o ligh is shown unde each s ain name. The
ini ials desc ibe each Neu ospo a species: Nc Neu ospo a c assa,N
Neu ospo a e aspe ma, and Nd Neu ospo a disc e a.
doi:10.1371/jou nal.pone.0033658.g004
The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 6 Ma ch 2012 | Volume 7 | Issue 3 | e33658
isola ed as a no h as Alaska [8]. Many Neu ospo a wild- ype
s ains ha e been collec ed in loca ions a ound he wo ld [6] bu
hei ca o enoid accumula ion o hei esis ance o UV adia ion
ha e no been cha ac e ized. A comp ehensi e in es iga ion o he
ela ionship be ween ca o enoid accumula ion, UV esis ance, and
la i ude will equi e ex ensi e sampling o he Neu ospo a popula-
ions in di e en loca ions. These expe imen s will help o assess i
he co ela ion be ween ca o enoid accumula ion and la i ude ha
we ha e unco e ed o he Neu ospo a popula ions in some a eas o
Spain a e obse ed in a mo e global scale.
Figu e 5. A nega i e co ela ion be ween ca o enoid accumula ion and la i ude. Mycelia om each wild- ype s ain we e exposed o ligh
du ing one day and he amoun o ca o enoids measu ed. Each poin ep esen s he a e age amoun o ca o enoids ob ained in wo independen
expe imen s and he la i ude o he si e whe e he wild- ype s ain was collec ed. The line shows he lineal eg ession o he 150 wild- ype s ains
cha ac e ized (coe icien o de e mina ion R
2
= 0.71).
doi:10.1371/jou nal.pone.0033658.g005
Figu e 6. Su i al o
Neu ospo a
conidia o UV adia ion. Conidia we e exposed o UV adia ion du ing di e en imes o kep uni adia ed as a
con ol. Cell iabili y was assayed a e pla ing i adia ed and uni adia ed conidia, coun ing he numbe o colonies a e 2–3 days o g ow h, and
compa ing he numbe o colonies ob ained wi h i adia ed and uni adia ed conidia. The plo shows he a e age and s anda d e o o he mean o
he pe cen age o su i al o UV in h ee independen expe imen s. The amoun o ca o enoids accumula ed by mycelia om each wild- ype s ain in
cul u es exposed o ligh is shown o e each s ain name. The colo used o symbols and lines iden i y each Neu ospo a species. The la i udes and
longi udes o he isola ion si es o each s ain a e he ollowing: C11A (42.74u,28.62u), PO10A (42.46u,28.50u), GC4-1A (27.88u,215.56u), PO9C
(42.46u,28.50u), GC4-4C (27.88u,215.56u), TF2-6A (28.37u,216.65u).
doi:10.1371/jou nal.pone.0033658.g006
The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 7 Ma ch 2012 | Volume 7 | Issue 3 | e33658
Ma e ials and Me hods
E hics s a emen
No speci ic pe mi s we e equi ed o his ield s udy. In
addi ion, no speci ic pe mission was equi ed as he samples we e
aken om public a eas and he s udy did no in ol e an
endange ed o p o ec ed species.
Collec ion and cul u ing o Neu ospo a wild ypes
We made wo ield ips o i e si es loca ed in opposi es ends o
Spain ha had su e ed an unusual high equency o i es du ing
he summe s o 2006 and 2007. The i s ip ook place du ing
Oc obe 2006 o isi Galicia a he no hwes e n co ne o he
Ibe ian Peninsula. The second ip ook place du ing Sep embe o
2007 o isi he Cana y Islands a he no hwes e n coas o
A ica. Addi ional samples we e aken in he p o ince o Ca´ce es
while a elling o Galicia. Mos o he si es we e su eyed 4–6
weeks a e he end o he i e. All he samples we e aken om
colonies g owing on he su ace o bu ned ege a ion bu we did
no pay a en ion o he o ien a ion ela i e o he sun o he
sampling si e. The loca ion o each sampling si e is desc ibed in
Table 1.
We ollowed s anda d me hods o handling wild ype isola es o
Neu ospo a, including collec ing, ini ial cul u ing, subcul u ing o
single conidia, and s o age [8]. A ield sample o conidia was
collec ed om a spo ula ing colony on o s e ile il e pape , which
hen was placed in a s e ile en elope. One colony pe plan was
sampled o up o 33 isola es pe si e. In addi ion, whe e possible,
up o ou isola es om he same plan we e collec ed pe si e. All
he wild ype s ains ha e been deposi ed in he Fungal Gene ic
S ock Cen e (FGSC; h p://www. gsc.ne ) unde accession
numbe s 10264–10289, 10461–10585 (Table S1).
We used he s anda d Neu ospo a c assa wild- ype s ain 74-
OR23-1VA (FGSC 2489 ma A). All s ains we e main ained by
g ow h in Vogel’s minimal media wi h 1.5% suc ose as ca bon
sou ce. S ain manipula ion and g ow h media p epa a ion
ollowed s anda d p ocedu es and p o ocols [3]. See also, he
Neu ospo a p o ocol guide (h p://www. gsc.ne /Neu ospo a/Neu
ospo aP o ocolGuide.h m).
Iden i ica ion o Neu ospo a species by DNA sequence
compa isons
To al genomic DNA was isola ed om each wild- ype s ain
and h ee polymo phic egions (unlinked, noncoding loci ha lank
mic osa elli es named TMI, TML, and DMG) we e ampli ied by
PCR, pu i ied, and sequenced. We ampli ied and sequenced an
addi ional polymo phic egion (QMA) om he N. e aspe ma
s ains. The PCR condi ions and he sequence o p ime s o he
ampli ica ion o each polymo phic sequence ha e been desc ibed
p e iously [32]. The DNA sequences ha e been deposi ed in
GenBank wi h he ollowing accession numbe s: JN017932–
JN018056 (DMG), JN030769–JN030893 (TMI), JN048514–
JN048638 (TML), and JN084107–JN084123 (QMA).
The sequences o he h ee loci (TMI, TML, and DMG) om
he wild- ype s ains we e combined in o a single da ase o
phylogene ic analysis. Mic osa elli e sequences we e omi ed om
he analyses. In he alignmen we included he DNA sequences
om he h ee loci ob ained om a se o e e ence Neu ospo a
s ains ha included N. c assa,N. disc e a,N. e aspe ma,N. si ophila,
N. in e media, and he ecen ly iden i ied N. hispaniola,N. me zenbe gii,
and N. pe kinsii [32–36]. The sequences o each loci we e aligned
using Clus al X [52]. Phylogene ic analyses we e pe o med using
MEGA4 [53]. Phylogene ic econs uc ion was applied o he
h ee loci independen ly and hei combined alignmen . The
e olu iona y his o y was in e ed using he Maximum Likelihood
and Neighbo -Joining me hods, eliabili y o he b anches was
es ima ed using he boo s ap me hod (500 eplica es). The bes
nucleo ide subs i u ion model was in e ed using jModelTes [54].
We iden i ied he species o each Neu ospo a wild- ype s ain
using h ee me hods. The i s me hod elied on he es ablishmen
o e olu iona y ela ionships be ween he DNA sequences
ob ained om each wild- ype s ain and DNA sequences ob ained
om ep esen a i es o a ious Neu ospo a species based on he
combined analysis o he h ee loci. Iden i ica ion o Neu ospo a
species using his mul ilocus genealogical app oach has p o en
success ul [32–35].
The second me hod o iden i y each wild- ype s ain was o
sea ch he Neu ospo a DNA sequences in GenBank o sequences
simila o he h ee loci ha we cha ac e ized om each wild- ype
s ain (TMI, TML, and DMG) using he p og am Blas [55]
accessed h ough he NCBI web se e (h p://www.ncbi.nlm.nih.
go /). We used de aul pa ame e s o he Megablas algo i hm ha
is op imized o highly simila sequences. The Neu ospo a species
ha ga e he bes hi a e each Megablas sea ch con i med he
species assignmen ob ained by phylogene ic compa isons. An
addi ional loci (QMA) was used o iden i y N. e aspe ma s ains
using Megablas .
The hi d me hod is based on he Phylogene ic Species
Recogni ion (PSR) concep desc ibed in [36]. A phylogene ic
species is ecognized i i sa is ied ei he o wo c i e ia: (1)
Genealogical conco dance: he clade was p esen in he majo i y
(2/3) o he single-locus Maximum-Likelihood genealogies, as
e ealed by a majo i y- ule consensus ee. (2) Genealogical
nondisco dance: he clade was well suppo ed in a leas one
single-locus Maximum-Likelihood genealogy, as judged by
boo s ap p opo ions and was no con adic ed in any o he
single-locus genealogy a he same le el o suppo . To iden i y
such clades, a ee possessing only b anches ha ecei ed a
boo s ap p opo ion .70% was chosen o ep esen each o he
h ee loci, hen a semis ic consensus ee was p oduced om
hese h ee ees. The semis ic ee was cons uc ed wi h he
p og am Componen [56].
To iden i y gene ic a ia ions be ween each Neu ospo a species
and among s ains isola ed om a single plan DNA sequences
om each locus wi h a simila i y highe han 95% we e g ouped
using he p og am UCLUST wi h de aul pa ame e s [57].
Ca o enoid analysis
App oxima ely 10
6
–10
7
conidia we e inocula ed in o 25 ml o
Vogel’s liquid minimal medium wi h 0.2% Tween 80 as we ing
agen , and placed in s e ile Pe i dishes. The pla es we e g own in
he da k o one day a 34uC and hen exposed o ligh unde a se
o luo escen bulbs (2 W/m
2
blue ligh ) a 22uC o one day o
kep in he da k as a con ol. Mycelia we e collec ed, ozen in
liquid ni ogen, and lyophilized. Ca o enoids we e ex ac ed om
0.1 g d y weigh samples as desc ibed [40]. To al ca o enoids we e
es ima ed om measu emen s o he maximal abso p ion spec a
in hexane, assuming an a e age maximal E (1 mg/l, 1 cm) = 200.
RNA pu i ica ion and quan i a i e RT-PCR
App oxima ely 10
5
conidia we e inocula ed in o 25 ml o
Vogel’s liquid minimal medium wi h 0.2% Tween 80 as we ing
agen , and placed in s e ile Pe i dishes. The pla es we e incuba ed
in he da k o wo days a 22uC and hen exposed o ligh unde a
se o luo escen bulbs (2 W/m
2
blue ligh ) a 22uC o 30 min o
kep in he da k as a con ol. Mycelia we e collec ed, ozen in
liquid ni ogen, and s o ed a 280uC. Fo RNA ex ac ion mycelia
we e dis up ed by wo 0.5-min pulses in a cell homogenize
The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 8 Ma ch 2012 | Volume 7 | Issue 3 | e33658
(Fas P ep-24, MP Biomedicals), in RNA ex ac ion bu e , wi h
1.5 g o zi conium beads (0.5 mm diame e ) in 1.9-ml sc ew-cap
ubes. The samples we e cooled on ice o 5 min a e he i s
pulse. The ex ac s in sc ew-cap ubes we e cla i ied by
cen i uga ion in a mic ocen i uge (13,000 pm) o 5 min p io
o RNA pu i ica ion. To al RNA om mycelia was ob ained using
he RNeasy Plan Mini Ki (Qiagen). Quan i a i e PCR
expe imen s we e pe o med o de e mine ela i e mRNA
abundance using one-s ep RT-PCR, using 25 ml Powe SYBR
G een PCR Mas e Mix (Applied Biosys ems), 6.25 U Mul iSc ibe
Re e se T ansc ip ase (Applied Biosys ems), 1.25 U RNase
Inhibi o (Applied Biosys ems), 0.2 mM o each p ime (al1F 59-
CCATGTACATGGGCATGAGC-39, al1R 59-AGATACCCT-
CGGCCAACTCC-39, al2F 59-CCATCGGATCGGGGACGA-
AG-39, al2R 59-CCAGGAAGAACGTGGCTTCT-39, ub-2F 59-
CCCGCGGTCTCAAGATGT-39, ub-2R 59-CGCTTGAAG-
AGCTCCTGGAT-39) and 50 ng o RNA. Quan i a i e PCR
analyses we e pe o med using a 7500 Real Time PCR Sys em
(Applied Biosys ems). The eac ion included e o ansc ip ion
(30 min a 48u), dena u a ion (10 min a 95u), and 40 PCR cycles
(15 sec a 95u, and 1 min a 60u). The esul s o each gene we e
no malized o he co esponding esul s ob ained wi h ub-2 o
co ec o sampling e o s. Then, he esul s ob ained wi h each
sample we e no malized o he RNA sample ob ained om each
mycelia kep in he da k.
Su i al o Neu ospo a conidia o UV adia ion
Conidia om each Neu ospo a s ain (400 ml, 10
5
conidia/ml)
we e placed in a s e ile Pe i pla e wi hou he co e and exposed
o UV adia ion om a lamp (Philips TUV 15W/G15 T8
Longli e, emission o sho -wa e UV adia ion wi h a peak a
253.7 nm) du ing di e en imes. A e UV exposu e conidia we e
dilu ed and inocula ed in pla es wi h FGS aga ha p omo es
colonial g ow h. All he manipula ions we e pe o med in dim
ligh o p e en pho o eac i a ion. The pla es we e incuba ed in
he da k a 22uC du ing 2–3 days and colonies we e coun ed. The
pe cen age o su i al o UV was ob ained om he numbe o
colonies a e each UV ea men compa ed wi h he numbe o
colonies in con ol pla es wi h uni adia ed conidia.
Suppo ing In o ma ion
Table S1 Wild- ype s ains o Neu ospo a om Spain.
(XLS)
Table S2 A wo-way analysis o a iance (ANOVA) be ween
Neu ospo a species, la i ude, and ca o enoid accumula ion.
(DOCX)
Acknowledgmen s
We hank Viole a Dı
´az Sa´nchez o he help wi h he quan i a i e RT-
PCR expe imen s. We hank Ma ha Me ow and Da id Jacobson o
hei expe ad ice. We hank he la e Da id Pe kins o his gene ous
suppo and encou agemen o pu sue Neu ospo a esea ch and o collec
Neu ospo a s ains in na u e. This a icle is dedica ed o he memo y o
C is obal Luque Lemos (1926–2007) and Luis Co ochano Leal (1931–
2010).
Au ho Con ibu ions
Concei ed and designed he expe imen s: LMC. Pe o med he expe i-
men s: EML LNS MO JRR. Analyzed he da a: EML GG LMC. W o e
he pape : LMC. Collec ed Neu ospo a wild- ype s ains in ield ips: LNS
MO JRR CRH VGT.
Re e ences
1. Da is RH, Pe kins DD (2002) Neu ospo a: a model o model mic obes. Na Re
Gene 3: 397–403.
2. Pe kins DD, Da is RH (2000) Neu ospo a a he millennium. Fungal Gene Biol
31: 153–167.
3. Da is RH (2000) Neu ospo a. Con ibu ions o a Model O ganism. Ox o d:
Ox o d Uni e si y P ess.
4. Selke EU (2011) Neu ospo a. Cu Biol 21: R139–140.
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The Fungus Neu ospo a in Spain
PLoS ONE | www.plosone.o g 9 Ma ch 2012 | Volume 7 | Issue 3 | e33658