A relationship between carotenoid accumulation and the distribution of species of the fungus neurospora in spain
Abstract
The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Ca´ceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88u and 42.74u. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.
Full text
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
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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
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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
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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.
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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