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ace elemen s and c and n iso ope
composi ion in wo mush oom
species om a mine-spill
con amina ed si e
Ma a Gil-Ma ínez1*, ca men M. na a o- e nández1, José M. Mu illo1, Ma ía . Domínguez2
& eodo o Ma añón1
ungi play a key ole in he unc ioning o soil in e es ial ecosys ems, and in pa icula in he
emedia ion o deg aded soils. he con ibu ion o ungi o ca bon and nu ien cycles, along wi h
hei capabili y o mobilise soil ace elemen s, is well-known. Howe e , he impo ance o li e his o y
s a egy o hese unc ions has no ye been ho oughly s udied. his s udy explo ed he soil- ungi
ela ionship o wo wild edible ungi, he ec omyco hizal Lacca ia lacca a and he sap o oph
Vol oplu eus gloiocephalus. ui ing bodies and su ounding soils in a mine-spill con amina ed a ea
we e analysed. iso ope analyses e ealed Lacca ia lacca a ui ing bodies we e 15n-en iched when
compa ed o Vol oplu eus gloiocephalus, likely due o he ans e o 15n-deple ed compounds o hei
hos plan . Mo eo e , Lacca ia lacca a ui ing bodies δ13c alues we e close o hos plan alues han
su ounding soil, while Vol oplu eus gloiocephalus ma ched he δ13c composi ion o ha o he soil.
ungal species p esen ed high bioaccumula ion and concen a ions o cd and cu in hei ui ing bodies.
Human consump ion o hese ui ing bodies may ep esen a oxicological isk due o hei ele a ed cd
concen a ions.
Fungi play a key ole in he unc ioning o e es ial ecosys ems due o hei di e se capabili ies and ele ance
in nu ien cycling. Fungal unc ionali y is di ec ly ela ed o hei wide li e his o y s a egies, being classi ied as
sap obes, pa asi es, pa hogens and/o symbion s. Sap obes (o sap o ophs) a e ee-li ing ungi ha ob ain nu i-
en s mos ly om decomposing soil o ganic ma e and, due o his abili y, a e conside edas he mos impo an
decompose s in e es ial ecosys ems. Symbio ic myco hizal ungi also play a key ole in nu ien cycling1,2.
Myco hizal ungi a e able o ake up nu ien s and wa e , and ans e hem o he hos plan , in exchange o
pho osyn ha es3,4. Ec omyco hizal ungi may also mobilise soil ace elemen s enhancing hei ans e om soil
o plan and/o ungal issues5. Sap o ophic ungi domina e he li e laye in soils and acqui e C and nu ien s
in he o m o o ganic subs ances by enzyma ic decomposi ion6. Symbio ic ec omyco hizal ungi a e no mally
ound in deepe soil laye s (well-deg aded li e and humus) wi h a high nu ien con en bu low C con en , as
hey a e no C-limi ed7,8. Ec omyco hizal ungi a e highly ele an in o es s, including Medi e anean ones,
as hey associa e wi h nume ous ee and sh ub species9. P e ious s udies ound independen oles be ween
ec omyco hizal and sap o ophic ungi, wi h ec omyco hizal ungi being able o deg ade ecalci an N- ich
compounds, while sap o ophic ungi being able o deg ade labile C- ich biopolyme s10. Al hough ec omyco -
hizal ungi ha e a lowe abili y o decompose o ganic ma e han sap obes, hey can ob ain esou ces along he
bio ophy–sap o ophy con inuum11,12. Fo example, limi ed supply o plan pho osyn ha e may inc ease enzyme
p oduc ion o ob aining labile ca bohyd a es om soil o ganic ma e by ec omyco izal ungi9,13. Ecological
consequences may a ise depending on he si ua ion o he ungus wi hin he con inuum as, o example, ec o-
myco hizal ungi uc i ica ion may ail i sepa a ed om plan oo s11,14. The e o e, he spec um o li e his o y
s a egies implies ha ungi di e in hei oles and in hei e ec s on ecosys em unc ioning.
Di e en mechanisms o esou ce acquisi ion, loss and cycling in ungi can be in e ed by s udying s able iso-
opes in ungal ui ing bodies (mush ooms)15–17. P e ious s udies ound ha ec omyco hizal ungal spo oca ps
1Depa amen o de P o ección del Sis ema Suelo, Plan a, Agua. Ins i u o de Recu sos Na u ales y Ag obiología de
Se illa, CSIC, Se ille, Spain. 2Depa amen o de C is alog a ía, Mine alogía y Química Ag ícola. Uni e sidad de Se illa,
Se ille, Spain. *email: [email p o ec ed]
open
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p esen highe δ15N alues and lowe δ13C alues han sap o ophic ones1,17–19. Ec omyco hizal ungi compe e
o N agains sap o ophic ungi and bac e ia, and hey disc imina e s ongly agains 15N20. Ec omyco hizal
ungi a e known o hei N iso ope ac iona ion in he plan -soil sys em, as ungi e ain 15N-en iched N while
15N-deple ed N is ans e ed o he hos plan . The e o e, analysing and compa ing δ13C and δ15N alues in ui -
ing bodies and unde lying soils could help us o unde s and di e ences be ween sap o ophic and myco hizal
s a egies o esou ce acquisi ion1.
Fungi ha e been ex ensi ely s udied in ace elemen con amina ed a eas as hey a e able o de elop mecha-
nisms o ole a e high ace elemen concen a ions (see Bellion e al. 2006 o ec omyco hizal ungi21). Fungi
may educe he en y o ace elemen concen a ions in o cells by a ange o ex acellula mechanisms, such as
chela ion by exc e ed ligands, cell-wall binding and enhanced e lux. Despi e his, some amoun o ace elemen s
may s ill en e cells, bu hei oxici y can be educed by o he in acellula mechanisms, such as chela ion by
pep ides and subcellula compa men a ion22. In basidiomyce es, he mechanisms o esponse o ace elemen
con amina ion a e di e se and species dependen 22,23. Fo example, he esponse o Aga icus o coppe (Cu) is o
p oduce me allo hionein, while he esponse o cadmium (Cd) is o induce he p oduc ion o mycophospha in22.
Due o hese p o ec i e mechanisms o ungi, many s udies ha e ocused on analysing he accumula ion o
ace elemen s in o abo eg ound ungal biomass, as human consump ion o edible mush ooms causes conce n.
In some ace elemen con amina ed soils, a high accumula ion o ace elemen s has been ound in edible mush-
ooms g owing he e. In a sil e -mining a ea o Czech Republic, high concen a ions o ace elemen s we e
ound in edible mush ooms: 149 mg kg−1 d y ma e o Cd in Aga icus sil a icus, 12.9 mg kg−1 d y ma e o me -
cu y (Hg) in Lepis a nuda and 16.2 mg kg−1 d y ma e o lead (Pb) in Lycope don pe la um24. In ano he s udy, in
a smel e a ea o Aus ia, he collec ed mush ooms had ele a ed concen a ions o zinc (Zn) (up o 777 mg kg−1)
and Cd (up o 127 mg kg−1)25.
Besides he nume ous s udies published, he ela ionships be ween ace elemen concen a ions in soils and
in ungal ui ing bodies a e no always consis en and emain unclea . Some s udies in he li e a u e ha e ound
ha ungal species may beha e as bioindica o s o ace elemen s, as concen a ions in ungal ui ing bodies
and hei subs a es co ela ed26–28. On he o he hand, sepa a e s udies epo ed ha elemen al concen a ions
in ungi and soil did no co ela e; hey could ha e he abili y o exclude ace elemen s o , a he con a y, o
accumula e hem in o hei biomass o le els well abo e (bu no necessa ily co ela ed o) he co esponding
concen a ions in soil25,29. This con o e sy is also a ec ed by he soil ex ac ion me hod selec ed o de e mine
ace elemen concen a ion30. Mos o he ungal s udies used soil o al ac ions (including esidual una ailable
soil ac ions) ins ead o eadily o po en ially a ailable ac ions31–33. Howe e , he use o soil “a ailable” ac ions
o compa e wi h spo oca p concen a ions is ecommended25,34.
In o de o imp o e ou unde s anding o he soil- ungi ela ionship, he selec ion and analysis o he mul iple
ac o s ha may explain he mush oom ace elemen accumula ion pa e ns is equi ed. Fi s , in mul i-elemen
con amina ed subs a es, in e ac ions among ace elemen s in soil and ungal biomass may u he complica e
ou abili y o o esee bioaccumula ion pa e ns. In condi ions o high concen a ions o soil ace elemen s he e
may be a compe i i e in e ac ion be ween some elemen s, o example be ween Cd and Zn. Some ec omyco hizal
species ha e a p e e en ial up ake o Zn (essen ial elemen ), h ough a compe i i e inhibi ion o Cd up ake ( oxic
elemen )35,36. Second, he e a e many en i onmen al ac o s ela ed o ungal accumula ion o ace elemen s37.
Soil a iables, such as soil o ganic ma e , pH and i on (Fe), manganese (Mn) and aluminium (Al) oxides and
oxyhyd oxides (among o he s) may explain ungal ace elemen accumula ion, as hey ha e a p o ound in luence
on ace elemen a ailabili y in he soil solu ion. Thi d, ophic di e ences among sap o ophs and ec omyco -
hizal ungi can be ela ed wi h a ia ions in ace elemen s concen a ions in bo h ui ing bodies and unde lying
soils, due o hei di e en oles in soil o ganic ma e decomposi ion and C and nu ien cycles10. The e o e, he
concen a ion o ace elemen s in he soil is no he only de e minan on hei accumula ion pa e n in ungal
ui ing bodies, as he soil- ungi ela ionship is e y complex. The Bioconcen a ion Fac o (BCF) (concen a ion
in spo oca p di ided by concen a ion in soil) is commonly calcula ed o elucida e his in ica e ansloca ion o
ace elemen s om su ounding soil o ui ing bodies37,38. This BCF is he e o e a e y use ul a io which allows
us o unde s and how di e en ungal species, uc i ying in simila en i onmen al condi ions, may di e in ace
elemen up ake and accumula ion.
This s udy is o in e es wi hin he o e all con ex o soil emedia ion p ocesses and a ises om he necessi y
o moni o ing ace elemen concen a ions in edible mush ooms g owing in a con amina ed si e, as well as
o elucida e he di e en ial esponse o sap o ophic and ec omyco hizal ungi o soil con amina ion by ace
elemen s. We analysed he dynamics o nu ien s and ace elemen s in wo wild edible ungi species: Lacca ia
lacca a (Scop.) Cooke, which was associa ed wi h s one pine and g ey-lea ed cis us by ec omyco hizal symbiosis
in he s udy a ea, and Vol oplu eus gloiocephalus (DC.) Vizzini, Con u & Jus o, sap o ophic ungi, uc i ying in
g assland soils. In a ace elemen con amina ed and emedia ed ecosys em, we selec ed hese ungal species as
bo h we e edible and uc i ied in he s udy a ea in he same pe iod; in addi ion, hey ha e con as ing li e his o y
s a egy.
The speci ic objec i es o his s udy we e: (1) De e mining ungal nu i ional sou ces using C and N iso ope
composi ion; (2) De e mining he accumula ion capaci y o ace elemen s in ungal ui ing bodies and he
ela ions wi h soil p ope ies; (3) E alua ing he po en ial oxicological isk o ui ing bodies (mush ooms) o
human consump ion.
Resul s
Soil p ope ies. Soil p ope ies in he s udy a ea we e ela i ely homogenous o o al C and N, a ailable
P, o al Mg and some ace elemen s (Tables1 and 2). Howe e , he e we e signi ican di e ences be ween soils
unde nea h ui ing bodies o ec omyco hizal Lacca ia lacca a and sap o ophic Vol oplu eus gloiocephalus ungi
o o he soil a iables. Despi e he ac ha soil pH was acidic in he s udy a ea, Lacca ia lacca a unde lying soils
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had lowe pH (mean di e ence o 1.47 uni s) han Vol oplu eus gloiocephalus soils (Table1). A ailable K and o al
Ca we e signi ican ly di e en in he soils unde nea h bo h species, wi h highe concen a ions in Vol oplu eus
gloiocephalus unde lying soils (Table1).
Soil pseudo- o al a senic (As), Cu, Fe, nickel (Ni), Pb and sulphu (S) we e ela i ely homogeneous in he
s udy a ea. Howe e , pseudo- o al Cd, cobal (Co), Mn and Zn di e ed be ween ungal species habi a s, being
signi ican ly highe heconcen a ions in Vol oplu eus gloiocephalus unde lying soils (Table2). Soil “a ailable”
(CaCl2-ex ac ed) concen a ions o ace elemen s p esen ed a di e en end han pseudo- o al alues. Soil
unde nea h Lacca ia lacca a spo oca ps had signi ican ly highe CaCl2-ex ac ed concen a ions o almos all
ace elemen s (Cd, Co, Cu, Mn, Ni and Zn), excep o Fe, Pb and S concen a ions ha we e simila in bo h soils
(Table2).
ca bon and ni ogen iso opes in soil and ungal spo oca ps. Soil C con en was simila o bo h
ungal species, howe e C iso ope composi ion o hese soils was di e en ; Lacca ia lacca a su ounding soils
we e 13C-en iched compa ed o Vol oplu eus gloiocephalus 13C-deple ed su ounding soils (Table1 and Fig.1A).
Ec omyco hizal Lacca ia lacca a ungal spo oca ps we e 13C-deple ed ela i e o sap o ophic Vol oplu eus gloi-
ocephalus ungi (Fig.1A). Lacca ia lacca a ui ing bodies we e 13C-deple ed compa ed o hei soil ( = 4.24;
p < 0.001), whe eas Vol oplu eus gloiocephalus ui ing bodies we e no signi ican ly di e en o hei soil
( = −1.31; p = 0.208) (Fig.1A).
Soil p ope ies Lacca ia
lacca a Vol oplu eus
gloiocephalus p
pH 4.6 ± 0.2 6.1 ± 0.2 −7.00 <0.001
C o al (%) 0.901 ± 0.152 1.09 ± 0.12 −1.00 0.330
N o al (%) 0.169 ± 0.015 0.195 ± 0.010 −1.49 0.157
Pa ailable (mg kg−1) 11.5 ± 2.7 10.8 ± 0.8 1.22*0.249
Ka ailable (mg kg−1) 166 ± 16 272 ± 14 −5.07 <0.001
Ca o al (mg kg−1) 2127 ± 149 3469 ± 130 −6.79 <0.001
Mg o al (mg kg−1) 3014 ± 98 3122 ± 92 −0.81 0.428
Table 1. Main soil chemical p ope ies associa ed o Lacca ia lacca a and Vol oplu eus gloiocephalus ungal
species. Mean alues (±SE) o N = 10. S uden ’s - es s a is ic and p alue (in bold p < 0.05) a e shown. *Da a
ans o ma ion o no mali y o he esiduals.
Soil ace
elemen s Lacca ia lacca a Vol oplu eus
gloiocephalus BV (P 90) p
Pseudo- o al
As 142 ± 7 123 ± 15 157 1.12 0.282
Cd 0.630 ± 0.062 1.08 ± 0.05 −5.59 <0.001
Co 8.38 ± 0.47 11.2 ± 0.4 34 −4.54 <0.001
Cu 174 ± 13 185 ± 8 108 −0.73 0.475
Fe 37222 ± 1187 35973 ± 1902 0.56 0.586
Mn 301 ± 18 418 ± 17 −4.66 <0.001
Ni 14.7 ± 0.4 14.9 ± 0.4 62 −0.34 0.737
Pb 227 ± 14 204 ± 26 117 0.77 0.456
S 2421 ± 319 1769 ± 438 −2.07§0.058
Zn 193 ± 10 263 ± 11 134 −4.67 <0.001
CaCl2-ex ac able
Cd 0.142 ± 0.019 0.042 ± 0.008 4.91 <0.001
Co 0.145 ± 0.038 0.019 ± 0.005 4.51§<0.001
Cu 1.72 ± 0.70 0.253 ± 0.031 −4.68§<0.001
Fe 4.90 ± 0.42 4.30 ± 0.87 0.62 0.544
Mn 15.2 ± 1.9 4.52 ± 0.88 5.19 <0.001
Ni 0.354 ± 0.036 0.103 ± 0.015 6.45 <0.001
Pb 0.082 ± 0.015 0.083 ± 0.021 −0.06 0.957
S 135 ± 75 74.5 ± 53.2 62◊0.182
Zn 14.8 ± 1.9 3.12 ± 0.91 5.66 <0.001
Table 2. Soil ace elemen sand S (pseudo- o al andCaCl2-ex ac able) associa ed wi h Lacca ia lacca a and
Vol oplu eus gloiocephalus ungal species. Mean alues (±SE) in mg kg−1 o N = 10. S uden ’s - es s a is ic
and p alue (in bold p < 0.05). Backg ound alues (BV) a pe cen ile 90 (P 90) in Sou h Po uguese Zone
soils a dep h 0–20 cm in mg kg−1 a e indica ed83. §Da a ans o ma ion o no mali y o he esiduals. ◊Non-
pa ame ic Mann-Whi ney es (U s a is ic).
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Soil N con en was simila o bo h ungal species as well as hei N iso ope composi ion (Fig.1B). In con as
o δ13C alues, ec omyco hizal Lacca ia lacca a spo oca ps we e 15N-en iched in compa ison o sap o ophic
Vol oplu eus gloiocephalus spo oca ps. Ec omyco hizal Lacca ia lacca a spo oca ps we e 15N-en iched com-
pa ed o he su ounding soil ( = −2.99; p = 0.009), whe eas sap o ophic Vol oplu eus gloiocephalus ungi we e
15N-deple ed ( = 6.51; p < 0.001) (Fig.1B).
chemical composi ion o ungal spo oca ps. The e we e di e ences in nu ien concen a ions be ween
he ungal species. Vol oplu eus gloiocephalus ui ing bodies p esen ed signi ican ly highe con en s o N, P, K
and Mg compa ed o Lacca ia lacca a spo oca ps, whils his endency was he opposi e o Ca, wi h highe
concen a ions in Lacca ia lacca a ui ing bodies. The e was no signi ican di e ence be ween ungal species
ega ding C con en (Fig.2).
The wo ungal species had di e en pa e ns o accumula ion o mos o he ace elemen sand S (Fig.3),
wi h highe concen a ions o As, Co, Cu, Fe, Ni, Pb and Zn in Lacca ia lacca a ui ing bodies, and o Cd and S in
Vol oplu eus gloiocephalus ui ing bodies. No signi ican di e ences we e ound o Mn among ungal spo oca ps
o hese species.
Bioconcen a ion ac o s o ace elemen s. Cadmium BCF was high in bo h ungal species (means
o 5.09–8.49; Table3). Mean BCF alues o Co, Cu and Zn we e close o one (and maximum alues exceeded he
uni y) in he myco hizal Lacca ia lacca a, bu no in he sap o ophic Vol oplu eus gloiocephalus. The es o he
ace elemen s (As, Fe, Mn, Ni, Pb) and S had low BCF alues o bo h species.
Acco ding o he NMDS o dina ion, he soil a iables ha bes explained BCF a iabili y we e pH, o al Ca
and a ailable K (Fig.4). Vol oplu eus gloiocephalus spo oca ps uc i ied in soils wi h highe pH and highe Ca
and K concen a ions compa ed o Lacca ia lacca a spo oca ps. Two de ined clus e s sepa a ed he ungal species
wi h a highe BCF o Cd and S in Vol oplu eus gloiocephalus, while Lacca ia lacca a spo oca ps p esen ed highe
BCF o he es o he elemen s (Fig.4).
The ans e a io o ace elemen s om soil (CaCl2-ex ac ed ac ion) o ungal spo oca ps was e y high
o Cd and Cu, mode a e o Zn and Fe, low o Co, Mn and Ni, and negligible (<1) o S (Fig.S1). The ans e
a ios om soil o ungus we e highe in Vol oplu eus gloiocephalus, in pa icula o Cd (x12), Zn (x6) and Mn
(x5), wi h he excep ion o Fe which was highe (x2) in Lacca ia lacca a.
In gene al, Pea son´s co ela ion coe icien s be ween ace elemen concen a ions in ungal spo oca ps and hei
unde lying soils (CaCl2-ex ac ed alues) we e low (TableS1). Lacca ia lacca a had a signi ican co ela ion (p < 0.05)
wi h Co, and ma ginally signi ican co ela ions (p < 0.10) wi h As and Mn, and Vol oplu eus gloiocephalus wi h Fe.
oxici y isk. Calcula ions o he minimum daily in ake o he s udied ui ing bodies o each maximum
ole able in ake limi s o oxici y in humans p o ided weigh s o e 1 kg o esh weigh o bo h species o As, Cu,
Fe, Pb and Zn. Howe e , he consump ion o 0.069 kg esh weigh o Vol oplu eus gloiocephalus o 0.132 kg esh
weigh o Lacca ia lacca a would be enough o each he daily limi o Cd (TableS2). (All he calcula ions we e
based on a pe son o 70 kg bodyweigh ).
Discussion
Soil mic oo ganisms play a key ole in nu ien cycling, plan symbioses, o ganic ma e decomposi ion, and
o he ecosys em p ocesses39. T ace elemen con amina ed soils a e o conce n due o hei oxic e ec s on soil
mic obes40. In hese soils, ungal communi ies p o ide many ecosys em se ices o egula ion; in gene al,
imp o ing soil and wa e quali y, nu ien cycling, soil e ili y and ca bon seques a ion41. In pa icula , he
ns
***
0
1
2
3
4
5
6
Soil Spo oca p
15N (‰)
*
**
-20.0
-22.5
-25.0
-27.5
-30.0 Soil Spo oca p
δ13C (‰)
Lacca ia lacca a Vol oplu eus gloiocephalus
δ
AB
Figu e 1. (A) Ca bon and (B) Ni ogen iso ope composi ion in soil and spo oca ps o Lacca ia lacca a and
Vol oplu eus gloiocephalus ungal species. Ba s ep esen mean alues and e o ba s ep esen s anda d e o o
N = 10. P alues esul ing om S uden ’s - es a e indica ed (ns: non-signi ican ; p < 0.05*; p < 0.01**; p < 0.001***).
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“myco emedia ion” po en ial o s abilise ace elemen s in ungal issues can be conside ed asano he ecosys em
se ice. Thus, ungal mycelium sec e es ex acellula enzymes and acids o b eak downo ganic con aminan s
and has a high me al-binding capaci y playing a p omising ole in emedia ion o ace elemen s42. Howe e ,
om ungal mycelium, ace elemen s could be channelled o ui ing bodies implying a nu i ional po en ial
haza d due o mush oom consump ion42. F ui ing bodies (mush ooms) o mo e han 1100 ungal species a e used
wo ldwide o ood and medicines, hus deli e ing p o ision and cul u al se ices43. Wi h excep ion o poisonous
species o edible ungi wi h high concen a ion o po en ially oxic ace elemen s, which a e a haza d o human
heal h, and hen ep esen a cul u al “disse ice”41.
The la ge-scale phy o emedia ion plan wi hin he con amina ed a ea conside ed in his s udy included plan -
ing o na i e ees and sh ubs44. Wi h ime, plan ed ees modi ied he p ope ies o unde lying soil, o example,
pine ees induced soil acidi ica ion and, consequen ly, highe concen a ions o soluble ac ions o ace ele-
men s44,45. T ees also in luenced su ounding soil mic obial ac i i ies, compa ed wi h adjacen eeless pa ches46.
In his he e ogeneous landscape mosaic he wo s udied ungal species uc i ied in con as ing habi a s and soils:
Lacca ia lacca a was associa ed wi h hei hos pine ees while sap o ophic Vol oplu eus gloiocephalus was abun-
dan in he g asslands wi hou ees. Soils su ounding Vol oplu eus gloiocephalus spo oca ps p esen ed highe
pseudo- o al concen a ions o some elemen s (Cd, Co, Mn and Zn) han Lacca ia lacca a soils (unde pine ees).
These di e ences may be caused by he acidi ica ion p ocess which inc eases losses o ace elemen s om soil
due o enhanced leaching and ake up by ege a ion and auna47. Howe e , elemen s wi h low mobili y, such as As
and Pb, had simila concen a ions be ween soil ypes. The opposi e pa e n was ound o CaCl2-ex ac ed ace
elemen alues; mos o he ace elemen s we e ound in highe concen a ions in soils su ounding Lacca ia lac-
ca a spo oca ps unde pine ees p obably due o he pine-induced soil acidi ica ion and subsequen ace elemen
mobilisa ion, as al eady men ioned.
The di e ences in habi a and in li e his o y s a egy in luence he mine al nu i ion o bo h ungal species.
Lacca ia lacca a spo oca ps we e ound in soils close o hei hos s Pinus pinea and Cis us albidus, wi h which
hey de elop symbio ic ela ionships. The lowe nu ien concen a ion o he myco hizal spo oca ps, compa ed
wi h hose o Vol oplu eus gloiocephalus, could be explained by he symbio ic associa ion as pine and cis us may
ecei e pa o he nu ien s aken up by his ungi, such as N and P10,19. In con as , he sap o ophic Vol oplu eus
gloiocephalus, ound in g assland a eas domina ed by he baceous species, comple ely elies on soil sou ces o
nu i ion wi hou loss by ans e o o he o ganisms. The nu i ional quali y o bo h ungal spo oca ps was
wi hin he usual con en ange o wild g owing mush ooms, excep o Mg ha was lowe in bo h species48.
Con as ing li e his o y s a egy o he s udied ungal species in luences C and N cycles. Despi e simila soil C
con en , he obse ed di e ences in soil δ13C alues may e lec a di e en iso ope composi ion o di e en u n-
o e a es, and he p e e en ial use o 12C compa ed o 13C in biological p ocesses49. Ca bon iso ope composi ion
in Vol oplu eus gloiocephalus ui ing bodies ma ched he a ios o he C soil sou ce, acco ding o i s sap o ophic
na u e as li e decay ungi. In con as , Lacca ia lacca a ui ing bodies we e 13C-deple ed (−28.14‰) compa ed
Figu e 2. Nu ien s in Lacca ia lacca a and Vol oplu eus gloiocephalus spo oca ps. Ba s ep esen mean alues
and e o ba s ep esen s anda d e o o N = 10. P alues esul ing om S uden ’s - es a e indica ed (ns:
non-signi ican ; p < 0.01**; p < 0.001***).

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Figu e 3. T ace elemen sand S in Lacca ia lacca a and Vol oplu eus gloiocephalus spo oca ps. Ba s ep esen
mean alues and e o ba s ep esen s anda d e o o N = 10. S uden ’s - es s a is ic and p alue a e indica ed
(ns: non-signi ican ; p < 0.05*; p < 0.01**; p < 0.001***). α Da a ans o ma ion o no mali y o he esiduals. β
Non-pa ame ic Mann-Whi ney es (U s a is ic).
BCF Species Mean SD Min Max
As L0.0096 0.0040 0.002 0.015
V0.0044 0.0021 0.002 0.009
Cd L5.09 2.87 2.75 12.58
V8.49 3.34 4.98 13.66
Co L0.626 0.602 0.204 2.286
V0.034 0.042 0.001 0.138
Cu L0.795 0.217 0.462 1.186
V0.351 0.114 0.243 0.634
Fe L0.0057 0.0018 0.031 0.0079
V0.0015 0.0003 0.001 0.0021
Mn L0.107 0.119 0.049 0.442
V0.064 0.011 0.040 0.077
Ni L0.044 0.027 0.018 0.100
V0.018 0.007 0.006 0.028
Pb L0.0086 0.0021 0.006 0.012
V0.0040 0.0027 0.002 0.011
SL0.0001 <0.0001 0.0001 0.0002
V0.0003 0.0002 0.0001 0.0006
Zn L0.942 0.173 0.715 1.296
V0.432 0.066 0.351 0.561
Table 3. Bioconcen a ion ac o (BCF) o pseudo- o al ace elemen sand S in Lacca ia lacca a (L) and
Vol oplu eus gloiocephalus (V). Bold alues indica e BCF > 1.
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o soil (−26.49‰), e lec ing he iso ope composi ion o i s hos pho osyn ha e, which could be he main C
sou ce1,50. The di e ences in N iso ope composi ion be ween bo h ungal species we e e en mo e conspicuous,
whe e ec omyco hizal Lacca ia lacca a ui ing bodies we e 15N-en iched in compa ison o he su ounding
soil; po en ially because o he ans e o 15N-deple ed compounds o he symbio ic plan s1,18,51–55. Whe eas,
sap o ophic Vol oplu eus gloiocephalus ui ing bodies had much lowe 15N han Lacca ia lacca a, and we e
15N-deple ed in compa ison o he soil, despi e ha ing simila N iso ope composi ion in bo h soil ypes.
A p e ious s udy in Sweden ound ungi we e able o ake up and accumula e ace elemen s such as Cd, Cu
and Zn in bo h spo oca ps and mycelium (BCF om 1.9 o 8.8) in espec o bulk soil concen a ions56. In his
s udy, we ound ha he ungal up ake o ace elemen s is dependen on bo h he chemical elemen and he un-
gal species5. Low selec i i y o ungal anspo e s o essen ial elemen s, such as Ca and Zn, a ou ed he ans-
po o oxic ions wi h simila chemical p ope ies, such as Cd22. In he s udy a ea, bo h Vol oplu eus gloiocephalus
and Lacca ia lacca a spo oca ps mainly bioconcen a ed Cd. P e ious s udies in he same a ea had ound ha
oo s o se en ee species also concen a ed Cd (BCF > 1), making hem sui able o he phy os abilisa ion o his
po en ially oxic elemen 44. Al hough Cd was accumula ed in ui ing bodies o bo h ungal species, he pa e ns
and ela ions wi h soil di e ed; Vol oplu eus gloiocephalus spo oca ps accumula ed much highe le els o Cd han
Lacca ia lacca a. Howe e , accumula ion o Ca and Zn we e opposi e, wi h highe concen a ions in Lacca ia lac-
ca a spo oca ps. The co ela ion be ween Cd le els in ui ing bodies and soils was posi i e o he sap o ophic
species bu nega i e o he ec omyco hizal one; a possible explana ion is ha Cd-Zn-Ca compe i ion in luence
elemen s accumula ion in hes udied spo oca ps57. A highe Ca and Zn con en was egis e ed in Lacca ia lacca a
spo oca ps, besides Vol oplu eus gloiocephalus su ounding soils eco ded ahighe Ca and Zn concen a ions.
The e o e, a compe i ion among Cd-Zn-Ca could be esponsible o he p e e ence o ansloca ion o Ca and Zn
o e Cd in Lacca ia lacca a ui ing bodies. On he o he hand, he p esence o Cd could induce he p oduc ion
o in acellula binding compounds such as me allo hioneins, cadys in, and phy ochela ins, and cellula com-
pa men aliza ion in his species22,58,59. Besides he an agonism o in e ac ion be ween chemical elemen s, he
bioconcen a ion o ace elemen s in ungal ui ing bodies is in luenced by soil p ope ies60. In his s udy, soil
pH, Ca and K we e ele an a iables explaining he BCF o ace elemen s in ui ing bodies, and hese a iables
we e posi i ely co ela ed among hem (p < 0.001). In acidic soils, he elease o mos ace elemen s inc eases,
being mo e a ailable in he soil solu ion and, he e o e, mo e accessible o mic oo ganisms61,62. In compa ison
wi h o he s udies, As concen a ions in Lacca ia lacca a spo oca ps a e b oad, depending on he loca ion and soil
condi ions, wi h a ange om 0.66 o 147 mg kg−1 in caps and s ipes63.
The CaCl2-ex ac ed ac ion o mos o he ace elemen s in he soil usually ep esen s a small p opo ion
o hei o al concen a ion (Table2). The e o e, he ans e a ios o ace elemen s om soil (CaCl2-ex ac ed
pool) o ungal spo oca ps a e unc ionally mo e ealis ic han BCF alues (calcula ed using pseudo- o al soil
concen a ions) and wo hwhile o conside . Thus, besides he impo an Cd accumula ion al eady discussed
abo e, we ha e de ec ed a high ans e o Cu om soil o ungus (o e 200 imes). O he ace elemen s wi h
ema kable soil o ungus ans e we e Zn (78 imes mo e in Vol oplu eus gloiocephalus) and Fe (48 imes mo e
in Lacca ia lacca a). I we assume ha spo oca ps a e e lec ing mycelium chemical composi ion, we can in e
ha bo h ungal species a e con ibu ing po en ially o he “mycos abilisa ion” o Cd, Cu, Zn and Fe in hese
con amina ed and emedia ed soils37,42.
The well-known capaci y o mush ooms o accumula e ace elemen s could make hem use ul o ganisms o
bioindica e soil con amina ion64. Howe e , in his s udy, ace elemen concen a ions in ui ing bodies we e no
signi ican ly co ela ed o soil CaCl2-ex ac ed concen a ions, wi h he excep ion o Co in Lacca ia lacca a. Many
s udies ha e also ound ha ui ing bodies a e no co ela ed wi h soil con amina ion by ace elemen s; he e o e
Figu e 4. Nonme ic mul idimensional scaling (NMDS) o dina ion o ace elemen bioconcen a ion ac o s
(BCF) o Lacca ia lacca a (o ange squa es) and Vol oplu eus gloiocephalus (whi e squa es) ungal species.
Black do s ep esen BCF o each ace elemen and S. B own a ows ep esen soil a iables wi h a signi ican
co ela ion wi h BCF (p = 0.001 o pH and K, p = 0.003 o Ca).
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concluding ha hey a e no good bioindica o s o soil ace elemen con amina ion32,65–67. On he con a y, he e
a e au ho s suppo ing he use o ungal spo oca ps as bioindica o s26–28. In ou s udy si e, he concen a ion o
ace elemen s in lea es o popla ees and in soil we e co ela ed o Cd, Cu, Mn and Zn68. The po en ial u ili y
as bioindica o would hen depend on he ungal species, he pa icula ace elemen , and he en i onmen al
condi ions.
F om he ood quali y pe spec i e, he usually high accumula ion o po en ially oxic elemen s in ungal ui -
ing bodies is a ele an ecosys em disse ice. T ace elemen s concen a ions in ui ing bodies we e simila o
hose measu ed in edible mush ooms om unpollu ed a eas67, excep o he inc eased mean concen a ions o
Cd in Vol oplu eus gloiocephalus (and maximum in Lacca ia lacca a) and mean Cu and maximum Fe, Mn and Zn
concen a ions in Lacca ia lacca a spo oca ps (TableS3).
Food egula ion o a oid oxici y isk includes concen a ion limi s o se e al ace elemen s in wild edi-
ble mush ooms. Thus, he Eu opean Union egula ion es ablished a concen a ion limi o 0.3 mg Pb kg−1 esh
weigh and 0.2 mg Cd kg−1 esh weigh o cul i a ed ungal species (Aga icus bispo us, Pleu o us os ea us and
Len inula edodes). Fo o he ungal species, his limi was es ablished a 1.00 mg Cd kg−1 esh weigh and no egu-
la ion limi was es ablished in o he species o Pb69,70. The maximum Cd concen a ion egis e ed in Vol oplu eus
gloiocephalus spo oca ps was 0.84 mg Cd kg−1 esh weigh , which is well abo e he Eu opean Union egula ion
limi o cul i a ed mush ooms, al hough s ill below he limi o wild species. The maximum Pb concen a ion
in ou spo oca ps was 0.25 mg Pb kg−1 esh weigh , which is also below he limi ( o cul i a ed species only).
The e o e, acco ding o Eu opean Union egula ion, nei he Cd no Pb concen a ions eached hese maximum
le els (TableS3; d y weigh ).
The Wo ld Heal h O ganiza ion71 e alua ed ce ain ace elemen con aminan s in ood o es ima e he ol-
e able in ake limi s (TableS2; PMTDI). We calcula ed he minimum weigh o each ole able limi s, and Cd
p esen ed he lowes weigh (TableS2). A daily in ake o 0.069 kg esh weigh o Vol oplu eus gloiocephalus o
0.132 kg esh weigh o Lacca ia lacca a ( o a pe son wi h 70 kg bodyweigh ) would each he maximal ole -
able Cd daily limi , wi hou aking in o accoun o he Cd daily sou ces. The consump ion o hese ungi could
ep esen a oxicological isk due o ele a ed Cd con en , especially Vol oplu eus gloiocephalus species, as his
elemen is o high oxicological impo ance o humans67,72. Lacca ia lacca a consump ion would en ail a highe
oxicological isk han Vol oplu eus gloiocephalus o he es o ace elemen s (As, Cu, Fe, Pb and Zn), howe e
he daily in ake o each oxici y (o e 1 kg esh weigh ) ep esen s an un ealis ically high le el o consump ion.
In any case, o a comple e e alua ion o ac ual po en ial oxici y o humans he e ec o mush oom p ocessing
du ing cooking on i s me al concen a ions should also be aken in o accoun .
conclusions
A e a long- e m es o a ion p ocess o mi iga e he e ec s o a mine-spill in SW Spain, Lacca ia lacca a and
Vol oplu eus gloiocephalus spo oca ps and su ounding soil analyses demons a ed ace elemen pe sis ence
in he s udy a ea. Some soil ace elemen concen a ions we e abo e backg ound alues and soil acidi ica ion
seemed o be esponsible o he highe ace elemen concen a ions in Lacca ia lacca a ui ing bodies. Analyses
o δ15N and δ13C in ui ing bodies and su ounding soils allowed us o di e en ia e he sap o ophic Vol oplu eus
gloiocephalus ungi om he ec omyco hizal Lacca ia lacca a ungi, p o ing his echnique as adequa e o di e -
en ia e ungal li e his o y and o con ibu e o a be e knowledge o he soil- ungi ela ions.
Bioconcen a ion ac o s demons a ed ha bo h ungal species accumula ed Cd, and ha Cu was ans e ed
om he soil “a ailable” (CaCl2-ex ac ed) pool o ungal spo oca ps. Human consump ion o Lacca ia lacca a
and/ o Vol oplu eus gloiocephalus spo oca ps collec ed in he a ea may ep esen a oxicological isk due o he
ele a ed concen a ions o Cd. The e o e, s ic con ol is equi ed in he a ea o a oid human consump ion o
hese ui ing bodies. Due o he abili y o hese species o accumula e some me als, moni o ing on ace elemen
concen a ionsin hese ungi is hus highly ecommended, e en i mobili y o me als in soils is assumed o be low.
Ma e ials and me hods
S udy a ea. The s udy was conduc ed in he Guadiama G een Co ido (Se ille, SW Spain; 37°23.165′N,
6°13.668′W) which is a public a ea managed by he Spanish Regional Go e nmen . The s udy a ea is loca ed
12 km downs eam om he Aznalcólla ailings dam ailu e, which a ec ed abou 4000 ha and a 60 km s e ch
along he Guadiama Ri e 73,74. Aznalcólla mine is loca ed in he Ibe ian Py i e Bel o med by a la ge concen a-
ion o polyme allic massi e sulphides wi h high g ade on sil e (Ag), Pb and Zn74. The mine-spill co e ed hese
soils wi h 6 hm3 o acidic wa e s and sludge loaded wi h a mix u e o ace elemen s, mainly As, Cd, Cu, Pb, hal-
lium (Tl) and Zn75. Since he acciden (in 1998) un il 2001, a emedia ion plan was pu in place which consis ed
in sludge and op soil emo al by hea y machine y and applica ion o amendmen s (suga lime, clay and o ganic
ma e ) o immobilise ace elemen s. Subsequen ly, a o es a ion o he a ea wi h na i e ees and sh ubs was ca -
ied ou 44. Di e ences in soil ype and emedia ion ou come c ea ed a he e ogeneous Co ido , and 15 yea s a e
he emedia ion o he a ea some pa ches s ill p esen ed high soil acidi ica ion and ace elemen con en 76. The
s udy a ea is one o hese deg aded a eas wi h high oxici y isks due o he na u ally sandy and acidic soil, wi h a
limi ed capaci y o soils o bu e agains acid d ainage caused by he emnan s o mining esidues.
S udy ungal species. The wo selec ed species in his s udy we e Lacca ia lacca a, which o m ec omyco -
hizae, and Vol oplu eus gloiocephalus, which is a sap obe (Fig.5). Bo h species belong o he Basidiomyco a di i-
sion and Aga icales o de , bu a e classi ied in di e en amilies. Lacca ia lacca a belongs o he Hydnangiaceae
amily and Vol oplu eus gloiocephalus o he Plu eaceae amily. Lacca ia lacca a is dis ibu ed b oadly in he
No he n Hemisphe e and has small, ed-o ange ui ing bodies. This ungal species has a wide ec omyco -
hizal hos po en ial, a low speci ici y and i is cha ac e is ic o young s ands o ees77. This species has been
iden i ied as componen o he ungal ec omyco hizal communi ies associa ed wi h Que cus ilex in he s udy
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a ea5,78. Vol oplu eus gloiocephalus is dis ibu ed in Eu ope and No h Ame ica and has bigge ui ing bodies
han Lacca ia lacca a wi h whi e o g ey-b own colou s79.
The specimens o ui ing bodies collec ed o his wo k we e egis e ed in he Uni e si y o Se ille He ba ium
o he codes SEV-C 51 o Lacca ia lacca a and SEV-C 50 o Vol oplu eus gloiocephalus. Molecula analyses we e
ca ied ou o con i m he species mo phological de e mina ion and o iden i y po en ial eco ypes. Sequences
a e a ailable in GenBank (h ps://www.ncbi.nlm.nih.go /genbank/) unde accession numbe s MK616245 -
MK616246 o Lacca ia lacca a and MK616344 - MK616347 o Vol oplu eus gloiocephalus.
Sampling design. In Decembe 2016, a ield sampling was conduc ed in o de o e alua e he wild g owing
edible ungal ui ing bodies ha uc i ied in he emedia ed soils o his a ea. F ui ing bodies we e sampled in
he loca ions whe e hey uc i ied, na u ally occu ing in sepa a ed habi a s. Lacca ia lacca a spo oca ps we e
ound close o he ec omyco hizal ee, Pinus pinea, and he sh ub, Cis us albidus. In con as , Vol oplu eus gloio-
cephalus spo oca ps we e ound in adjacen eeless a eas co e ed by di e en he baceous species. The e o e, sam-
pling loca ions we e dependen on he dis ibu ion o hese habi a s in he s udy a ea (Fig.S2). Fo each species,
en poin s and a leas h ee spo oca ps pe poin we e sampled. Soil su ounding each spo oca p was aken ( op
0–5 cm dep h) wi h an auge om a leas i e loca ions wi hin each poin o make a composi e sample pe poin .
Spo oca ps and soils we e kep e ige a ed un il being p ocessed he day a e sampling.
Soil analyses. Soil samples ( op 0–5 cm dep h; 100 g esh weigh ) we e ai -d ied and sie ed o <2 mm o
chemical analysis, whe e soil pH was measu ed in a 1:2.5 soil-wa e suspension a e 30 min o shaking. B ay 1
me hod was selec ed o de e mine soil P in hese acidic soils80. A ailable K was ex ac ed wi h 1 M ammonium
ace a e and de e mined by mul ipa ame ic B an-Luebbe au oanalyse . Pseudo- o al concen a ions o ace ele-
men s (As, Cd, Co, Cu, Fe, Mn, Ni, Pb and Zn), S, Ca and Mg we e ex ac ed by diges ion o soils, g ound o
<60 µm, wi h aqua egia (1:3 / conc. HNO3/ HCl) in a Digip ep MS block diges e (SPS Science) o 2 h a
110 °C. T ace elemen s (As, Cd, Co, Cu, Fe, Mn, Ni, Pb and Zn)and S we e ex ac ed om <2 mm sie ed soils
wi h a 0.01 M CaCl2 solu ion o es ima e hei “bioa ailabili y”81. Ex ac s a e diges ion wi h aqua egia o CaCl2,
we e de e mined by induc i ely coupled plasma op ical emission spec opho ome y (ICP-OES) wi h Va ian ICP
720-ES. The quali y o he analyses was assessed using he e e ence sample ERM-CC141 (loam soil) and eco e -
ies om 93.8% o 100.4% we e ob ained. A senic ex ac ed by CaCl2 p esen ed concen a ions below de ec ions
limi s (0.005 mg kg-1), and i s “a ailabili y” is unde es ima ed by his me hod.
chemical composi ion o ungal ui ing bodies. F ui ing bodies we e d y cleaned wi h a b ush
and esh weighed, wi h some biomass kep a −80 °C o u he species iden i ica ion by molecula analysis.
Spo oca ps we e d ied a 70 °C o 48 h and s o ed un il analysis. The whole ui ing body (cap, hymenopho e and
s alk) was analysed as one single sample, as all he pa s a e usually inges ed when consumed by humans. F ui ing
bodies mac onu ien s (Ca, K, Mg, S and P) and ace elemen s we e ex ac ed by diges ion wi h HNO3 in a
Digip ep MS block diges e (SPS Science) o 2 h a 110 °C and de e mined by ICP-OES wi h Va ian ICP 720-ES.
A senic was de e mined by ICP-OES wi h Ion Exchange Hyd ide Gene a ion (IE-HG-ICP-OES) me hodology82.
The quali y o he analysis was assessed using he e e ence sample ID 124 Type Luce ne (Medicago sa i um)
(WEPAL; IPE) o As, wi h eco e ies o 95.9%, and he e e ence sample INCT-OBTL-5 ( obacco lea es) o he
es o he ace elemen s, wi h eco e ies om 79.9% o 112.2%.
Figu e 5. F ui ing bodieso he ec omyco hizal Lacca ia lacca a (le ) o e needles o Pinus pinea, and o he
sap ophy ic Vol oplu eus gloiocephalus ( igh ) o e open g assland. Pho os we e aken in Decembe 2016 in he
Guadiama G een Co ido , SW Spain (au ho s: T. Ma añón and C. M. Na a o-Fe nández).