Fo es Ecology and Managemen 519 (2022) 120340
A ailable online 3 June 2022
0378-1127/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC license (h p://c ea i ecommons.o g/licenses/by-
nc/4.0/).
In luence o s and age and si e condi ions on ec omyco hizal ungal
dynamics in Cis us ladani e -domina ed sc ubland ecosys ems
Pablo Ma ín-Pin o
a
,
*
, Juan And ´
es O ia-de-Rueda
a
, Ta ek Dejene
a
,
b
, Olaya Media illa
a
,
c
,
Ma ía He n´
andez-Rod íguez
a
,
c
, Jos´
e A. Reque
a
, Ignacio Sanz-Beni o
a
, Ma ía San os
a
,
J´
ozse Geml
d
a
Sus ainable Fo es Managemen Resea ch Ins i u e UVa-INIA, A enida Mad id, s/n, 34004 Palencia, Spain
b
E hiopian En i onmen and Fo es Resea ch Ins i u e (EEFRI), P.O. Box 30708 Code 1000, Addis Ababa, E hiopia
c
IDFo es -Bio ecnología Fo es al Aplicada, Calle Cu ido es 17, 34004 Palencia, Spain
d
MTA-EKE Lendüle En i onmen al Mic obiome Resea ch G oup, Esz e h´
azy K´
a oly Uni e si y, Le´
anyka u. 6, H-3300 Ege , Hunga y
ARTICLE INFO
Keywo ds:
Cis us ladani e
Clima e egion
Di e si y
Fungi
Me aba coding
Phospho us
pH
S and age
ABSTRACT
Cis us ladani e -domina ed ecosys ems a e widely dis ibu ed in he Wes e n Medi e anean basin and a e
a ec ed by ecu en i es. Al hough hese sc ublands we e adi ionally conside ed unp oduc i e, hese sys ems
p o ide signi ican ecological bene i s ia mush oom p oduc ion, which has inc eased in e es in be e unde -
s anding hese ecosys ems o es o e and p omo e p oduc i i y. We analyzed 48 plo s loca ed in Sup a- and
MesoMedi e anean egions in wes e n Spain o assess he soil ungal communi y and hei ecological d i e s
using ITS2 DNA Illumina Miseq. The s udy plo s comp ised young (ea ly-s age successional s ands), middle-
(middle-s age successional s ands), and la e-s age s ands. Shannon di e si y index alues o o al ungi we e
highe unde a MesoMedi e anean han unde a Sup aMedi e anean clima e ype, whe eas he ichness alues
o ec omyco hizal (EcM) axa we e highe in la e-s age s ands han in he younge s ands. EcM communi y
composi ion was in luenced by s and age, clima ic a iables and edaphic pa ame e s. These C. ladani e -
domina ed ecosys ems suppo di e se ungi, including edible species such as Bole us, Lac a ius, and Lacca ia,
unde speci ic p ecipi a ion, empe a u e, and la e-s age s and ac o condi ions. Al hough o es managemen
canno modi y empe a u e and p ecipi a ion, managemen s a egies ha conside mosaic landscapes o educe
he se e i y o po en ial i es and ha e ain la e successional s ands could p o ide sui able habi a s o p o-
mo ing ungal di e si y, p oduc ion, and unc ion in hese sc ubland sys ems.
1. In oduc ion
Sc ublands domina ed by Cis us ladani e L. a e dis ibu ed
h oughou he Wes e n Medi e anean basin (O ia De Rueda e al.,
2008; Quin ela-Saba ís e al., 2011) owing o hei his o ical adap a ion
o ecu en i es in his egion (Quin ela-Saba ís e al., 2011). Li le
a en ion has been gi en o he managemen o Cis us-domina ed
sc ublands because hey ha e been deemed unp oduc i e due o hei
poo soils (Ma ín-Pin o e al., 2006; O ia De Rueda e al., 2008).
Howe e , p e ious s udies ha e shown ha hese Cis us-domina ed
sc ublands can p o ide bene i s in he o m o wild edible mush ooms
when p ope ly managed (He n´
andez-Rod íguez e al., 2015a). The mos
aluable ungal species associa ed wi h his sys em is Bole us edulis,
which is a highly p ized gas onomic mush oom in many coun ies (Boa,
2004). Indeed, in 2014, B. edulis yields o o e 271.10 kg ha
−1
we e
eco ded (Alday e al., 2017). In addi ion o hei economic ole,
C. ladani e -domina ed sc ublands play an impo an ecological ole in
he eco e y o o es s ands pos - i e by hos ing myco hizal ungi ha
a e hen able o colonize o he ee oo s as he new s and de elops
(He n´
andez-Rod íguez e al., 2015b). The apid pos - i e eco e y o
Cis us plan s is a o ed by he p esence o a ungal coho ha can su -
i e he dis u bance. Fu he mo e, symbio ic ne wo ks could play a key
ole in educing po en ial soil e osion (Busca do e al., 2012) in he i s
ain ollowing a i e in his ecosys em.
As an ecological ac o , i e in gene al a ec s ungal communi y
composi ion (Cai ney and Bas ias, 2007) and dec eases spo oca p p o-
duc ion (He n´
andez-Rod íguez e al., 2013) and he abundance o
ec omyco hizal (EcM) ungi (Cas a˜
no e al., 2020). In addi ion o he
* Co esponding au ho a : Depa men o Vege al P oduc ion and Na u al Resou ces, Uni e si y o Valladolid, A enida Mad id, s/n, 34004 Palencia, Spain.
E-mail add ess: [email p o ec ed] (P. Ma ín-Pin o).
Con en s lis s a ailable a ScienceDi ec
Fo es Ecology and Managemen
jou nal homepage: www.else ie .com/loca e/ o eco
h ps://doi.o g/10.1016/j. o eco.2022.120340
Recei ed 14 Feb ua y 2022; Recei ed in e ised o m 26 May 2022; Accep ed 29 May 2022
Fo es Ecology and Managemen 519 (2022) 120340
2
hos plan ’s succession, o he ac o s such as clima e and edaphic pa-
ame e s ha e been shown o a ec ungal communi ies and hei dis-
ibu ion (de V ies e al., 2012; Ye geau e al., 2009). Hos plan
succession a ec s he soil ungal communi y owing o changes in he
hos ’s esou ce equi emen and he soil ma ix a ailabili y (Tian e al.,
2017). Fo example, he g ow h o he hos o e ime is concu en wi h
a dec ease in soil e ili y, as ni ogen and phospho us become limi ing
ac o s (Cas a˜
no e al., 2019). This same p ocess occu s du ing second-
a y succession, which ollows a dis u bance e en . Se e e dis u bance
leads o dis inc e ec s on ungal communi ies because i can p o ide
open space o he su i al o di e en ungal species (Chen e al., 2018;
Shi e al., 2019) and elease nu ien s o suppo he g ow h o hese
species (Denslow, 1995). Thus, as we mo e along he plan succession,
ungal species composi ion changes (Toi anen e al., 2012), wi h
dis inc ungi colonizing dis inc successional s ages o he hos plan
(Toi anen e al., 2012), such as he EcM ungi (Smi h e al., 2002). Thus,
a shi om ea ly-s age EcM communi ies owa d la e-s age EcM com-
muni ies would e lec he s and condi ions, which can be explained in
e ms o he e ili y o he soil. Howe e , edaphic a iables can in lu-
ence nu ien a ailabili y, which is expec ed o change as he s and
de elops (Na a e al., 2003). Thus, he su i al and g ow h o he hos
plan could be suppo ed by he a ailabili y o di e en EcM species in
he soil, pa icula ly du ing he la e-s age succession o hos plan s, such
as C. ladani e s ands (He n´
andez-Rod íguez e al., 2013). In his ega d,
EcM colonizing species can also be di e en ia ed based on hei explo-
a ion dis ance a di e en s ages o hos de elopmen (Geml, 2019).
S and- eplacing wild i es a e a majo dis u bance in Medi e anean
ecosys ems. Wild i es pa icula ly a ec Cis us-domina ed sc ublands
(He n´
andez-Rod íguez e al., 2013; Sanz-Beni o e al., 2022) and can
al e bo h he ege a ion and soil p ope ies (G ogan e al., 2000),
mainly due o in ense hea ing and ash deposi ion (Peay e al., 2009).
Changes in he ege a ion and edaphic a iables may signi ican ly
impac soil ungal communi ies in hese widely dis ibu ed ecosys ems.
Thus, i e can a ec he subsequen s uc u e o ungal communi ies
ollowing succession pa e ns ha a e mainly d i en by he dynamics o
pos - i e plan communi ies (Cai ney and Bas ias, 2007) o C. ladani e
(He n´
andez-Rod íguez e al., 2013). As Cis us plan s g ow a e a i e,
changes occu in he succession o he associa ed ungal communi y
(Gassibe e al., 2011). Howe e , he in luence o hos plan succession
and en i onmen al a iables on soil ungal communi y composi ion in
C. ladani e -domina ed sc ublands ha e no ye been e alua ed. Mos o
he s udies conduc ed in hese ecosys ems ha e been pe o med a local
scales and in es iga ed a limi ed se o managemen a iables, such as
o al o pa ial clea ing and p esc ibed bu ning (Cas a˜
no e al., 2020;
He n´
andez-Rod íguez e al., 2013). Howe e , knowledge o soil ungal
communi ies is impo an o unde s and he ela ionship be ween
spo oca p p oduc ion le els o aluable species and he amoun o
mycelium in he soil (Media illa e al., 2017). To gain a deepe unde -
s anding o he di e en en i onmen al ac o s d i ing ungal commu-
ni ies as well as hos plan succession, we should conside a wide ange
o a iables om b oad geog aphic a eas. This should enable he
de elopmen o s a egies o manage mosaic landscapes o educe he
se e i y o po en ial i es in hese widely dis ibu ed ecosys ems.
In his s udy, we hypo hesized: (1) ha o al soil ungal di e si y
would be a ec ed by C. ladani e s and age, esul ing in a cumula i e
inc ease in ichness in la e-s age s ands. Fu he mo e, (2) we expec ed
ha he EcM ungal communi y would show changes along C. ladani e
s and age g adien s ha would be ela ed o di e ences in si e condi-
ions, such as clima e a iables and soil pa ame e s, unlike he sap o-
ophic axa. Finally, (3) we also expec ed ha he EcM ungi would be
di e en ia ed by hei explo a ion dis ance, wi h mo e abundan
ex a adical mycelium belonging o he sho -dis ance explo a ion- ype
o EcM in ma u e s ands, whe eas medium- o sho -dis ance explo a-
ion- ype EcM would no be a ec ed by C. ladani e s and age. To es
hese hypo heses, we in es iga ed he e ec o C. ladani e s and age,
clima e ype, and edaphic a iables on ungal communi ies p esen in
he soil. Ou speci ic objec i es we e, he e o e: (i) o unde s and how
he di e si y and ichness o he o al soil ungi and he EcM gene a a e
a ec ed by C. ladani e s and age a e i e; (ii) o iden i y clima e and
edaphic a iables ha signi ican ly d i e he composi ion o EcM and
o he ungal guilds.
2. Ma e ials and me hods
2.1. S udy a ea
The s udy a eas a e in he cen al-wes pa o Spain: in Luyego in he
p o ince o Le´
on (LE) and in Mayalde in he p o ince o Zamo a (ZA),
which ha e a Sup aMedi e anean he mo ype; and in Calzada de O o-
pesa in he p o ince o ´
A ila (AV) and in Aliseda in he p o ince o
C´
ace es (CC), which ha e a MesoMedi e anean he mo ype (Fig. 1).
The mo ype classi ica ion is mainly based on he win e empe a u es in
hese egions (Ri as-Ma ínez, 1983). The MesoMedi e anean he mo-
ype is de ined by win e empe a u es anging be ween 13 and 17 ◦C
and by equen os s in he win e , whe eas he summe is ho . Simi-
la ly, he Sup aMedi e anean he mo ype is also cha ac e ized by
empe a u es anging om 8 o 13 ◦C in win e . F os s and snow a e
common du ing he win e . Acco ding o he he mo ype classi ica ion
o Ri as-Ma ínez e al. (2011), bo h he Meso- and Sup aMedi e anean
s udy a eas a e cha ac e ized by a Medi e anean clima e (i.e., a d y
season o a leas wo mon hs in he summe , wi h mos o he p ecipi-
a ion alling be ween Oc obe and Feb ua y a all sampling si es. The
mean annual p ecipi a ion a he si es loca ed in he Sup a-
Medi e anean p o inces is 707 mm a Luyego (LE) and 464 mm a
Mayalde (ZA), while he mean annual empe a u es a e 9.5 ◦C and
11.3 ◦C, espec i ely. The mean annual p ecipi a ion a he si es loca ed
in he MesoMedi e anean p o inces is 677 mm a Calzada de O opesa
(AV) and 518 mm a Aliseda (CC), while he mean annual empe a u es
a e 14.9 ◦C and 16.1 ◦C, espec i ely. Clima ic a iables o all s udy
si es we e ob ained om nea by me eo ological s a ions.
The selec ed s udy a eas a e domina ed by Cis us ladani e ege a ion
and a e cha ac e ized by a wide ange o ele a ions anging om 300 m
abo e sea le el a he sampling si es in he sou h o 1080 m abo e sea
le el in he no h. Paleozoic me amo phic (sla e and qua zi e) and
plu onic (g ani e) ocks domina e he landscape, excep a he Mayalde
(ZA) si e, whe e Te ia y sands a e p edominan .
2.2. Expe imen al design, soil sampling, and molecula wo k
In May 2019, we es ablished plo s a he ou s udy si es o e alua e
he in luence o edaphic a iables and s and age condi ions on ungal
communi ies. The selec ed s udy si es comp ised h ee di e en s and
ages. In o ma ion ega ding he age o he s ands was ob ained om he
Regional Fo es Managemen Se ices. The age classes we e ea ly suc-
cessional s age s ands (4–6 yea s), middle-s age s ands (10–12 yea s),
and la e-s age successional s ands (20–22 yea s). Thus, ou expe imen al
design was composed o ou si es (AV, LE, CA, and CC), wi h ou blocks
pe si e. The blocks we e loca ed 2 o 3 km apa and he a ea o each
block anged om 1.5 o 2 ha. Each block comp ised a plo o each o he
h ee s and age g oups, wi h each plo measu ing 50 m ×50 m (i.e., 4
si es ×4 blocks pe si e ×3 sampling plo s pe block, i.e., 48 sampling
plo s in o al). Soil samples we e collec ed in June 2019. In o al, 15 soil
samples we e collec ed om each plo using an auge o a dep h o 20
cm, including he o ganic and mine al laye s. Soil samples we e pooled
pe plo o c ea e one composi e soil sample and o p ese e he he -
e ogenei y o he e ain. A e collec ion, samples we e d ied a oom
empe a u e un il a cons an weigh was ob ained and hen sie ed (1
mm “mesh”). A subsample o each plo was s o ed a –20 ◦C o mo-
lecula analysis. Chemical analyses we e pe o med acco ding o Spa ks
e al. (1996) o de e mine he soil pH (wa e -based), d y ma e (%),
o al phospho us (P), o al ca bon (C), and o al ni ogen (N) con en s o
he soil samples (Table 1).
P. Ma ín-Pin o e al.
Fo es Ecology and Managemen 519 (2022) 120340
3
2.3. Molecula analysis
We used he Qiagen Powe Lyze ® Powe Soil™ DNA Isola ion Ki
(MoBio Labo a o ies Inc., Ca lsbad, CA, USA) o ex ac DNA om 0.25 g
o soil pe sample acco ding o he manu ac u e ’s p o ocol. PCR am-
pli ica ions employed p ime se s ha a ge ed ITS4 and he ITS7. PCR
eac ions o each sample we e ca ied ou in iplica e o minimize PCR
biases. PCR eac ions we e pe o med in 20
μ
L eac ion olumes con-
aining 11.22
μ
L o Modi ied Quan iza ion (MQ) wa e , 1.60
μ
L o DNA
empla e, 2.00
μ
L o 10 ×bu e , 1.40
μ
L o MgCl
2
(50 mM), 1.60
μ
L o
dNTPs (10 mM), 0.50
μ
L o Bo ine Se um Albumin (2%), 0.80
μ
L o
e e se and o wa d p ime s (10
μ
M), and 0.08
μ
L o Pla inum Taq
polyme ase (In i ogen, Ca lsbad, CA, USA). The ollowing PCR condi-
ions we e used: an ini ial dena u a ion s ep a 95 ◦C o 5 min; hen 37
cycles o 95 ◦C o 20 s, 56 ◦C o 30 s, and 72 ◦C o 1.5 min; ending wi h
one cycle o 72 ◦C o 7 min. To ampli y he ITS2 DNA egion (ca. 250
bp), we used he o wa d p ime ITS7 (Ih ma k e al., 2012) and he
ba coded e e se p ime ITS4 (Whi e e al., 1990). Sample-speci ic
Mul iplex Iden i ica ion DNA ags we e used o label he ITS4 p ime .
Each se o PCR eplica es also included a nega i e con ol comp ising
MQ wa e ins ead o DNA ha unde wen PCR unde he same expe i-
men al condi ions and was shown o be amplicon- ee on a gel.
Sequencing was pe o med using an Illumina MiSeq pla o m (BaseClea
BV).
2.4. Bioin o ma ic analysis
We used cu adap (Ma in, 2011) o im low-quali y ends and
me ged he pai ed eads using USEARCH .10.0.240 (Edga , 2010).
Cu adap was se wi h a quali y sco e o 5 and a minimum sequence
leng h o 200 bp. P ime pai s (ITS4 and ITS7) we e immed and se-
quences wi h an expec ed e o o >1 we e emo ed. The emaining
sequences we e me ged in o unique sequence ypes on a pe -sample
basis using USEARCH .8.0 (Edga , 2010) while p ese ing ead
coun s. High-quali y sequences we e g ouped wi h USEARCH a 97%
sequence simila i y o gene a e ope a ional axonomic uni s (OTUs)
while simul aneously excluding sequences ep esen ing OTUs wi h
<70% simila i y o <200 bp pai wise alignmen leng h o a ungal
sequence. The sequences we e assigned o axonomic g oups based on
pai wise simila i y sea ches agains he cu a ed UNITE +INSD ungal
ITS sequence da abase ( e sion .8.0), which con ains iden i ied and
uniden i ied sequences assigned o species hypo hesis g oups de ined
based on dynamic sequence simila i y h esholds (K˜
oljalg e al., 2013).
Func ional g oups we e assigned o each OTU using Fungal T ai s
(P˜
olme e al., 2020). Fu he classi ica ion o EcM ungi as ei he sho -
dis ance explo a ion, long-dis ance explo a ion, o mixed-dis ance
explo a ion ypes was pe o med ollowing he c i e ia p oposed by
Geml (2019) and da a published by Age e (2006), Tede soo and Smi h
(2013), and he DEEMY da abase (h ps://deemy.de). Finally, edible
ungi we e iden i ied using specialis books and ungal guides, such as
Ge ha d e al. (2000) and Mo eno and Manj´
on (2010), o assess he
comme cial impo ance o he ungal communi ies in he s udy a eas.
2.5. S a is ical analysis
K ona cha s we e used o isualize he axonomic dis ibu ion o all
Fig. 1. A map o he ou sampling locali ies (indica ed by colo ed do s) in he cen al-wes pa o Spain.
Table 1
Mean chemical p ope ies o soils. Di e en lowe case le e s indica e signi ican di e ences among sampling si es, bioclima e ypes, o s and age based on LME and
Tukey’s HSD es . The numbe s in pa en heses a e he s anda d e o .
Soil pa ame e s Si es Bioclima e S and age
Le´
on Zamo a ´
A ila C´
ace es Sup a Meso Ea ly Middle La e
pH 4.61(5.49)b 4.96(5.72)a 5.11(6.24)a 4.95(5.65)a 4.74(5.63)a 5.02(5.23)b 4.92(5.61)a 4.76(5.49)a 4.76(5.51)a
P Olsen (mg/kg) 16.02(0.08)ab 5.28(0.20)b 23.95(3.29)a 5.23(0.08)b 11.12(0.95)a 15.42(2.69)a 9.46(0.84)a 16.61(2.86)a 12.25(1.79)a
% N 0.16(0.01)a 0.07(0.01)b 0.15(0.01)a 0.13(0.01)a 0.11(0.01)a 0.14(0.01)a 0.13(0.01)a 0.14(0.01)a 0.13(0.01)a
% C 2.48(0.14)a 0.95(0.05)b 2.64(0.14)a 2.05(0.10)a 1.71(0.15)a 2.35(0.13)b 1.90(0.12)a 2.12(0.15)a 2.23(0.16)a
% D y ma e 98.88(0.05)b 99.56(0.02)a 98.54(0.04)c 99.07(0.03)b 99.22(0.06)a 98.81(0.06)b 99.03 (0.06)a 99.01(0.06)a 99.00(0.07)a
No e: he Le´
on and Zamo a si es ha e a Sup aMedi e anean bioclima e (Sup a) while ´
A ila and C´
ace es ha e a MesoMedi e anean bioclima e (Meso). The pH alues
a e p o ided by he 95% Con idence in e als calcula ed om [H
+
] and hen back- ans o med o pH gi en ha pH =–log
10
[H
+
].
P. Ma ín-Pin o e al.
Fo es Ecology and Managemen 519 (2022) 120340
4
ungi and guilds based on OTU ichness ollowing Tede soo e al. (2020).
To no malize he OTU able o subsequen s a is ical analysis, we
a e ied he numbe o high-quali y ungal sequences (35,404 eads).
A e s anda dizing he en i onmen al a iables and ans o ming he
OTU a e ied ma ix using he Hellinge ans o ma ion me hod, ca-
nonical co espondence analysis (CCA) was pe o med o analyze he
ungal communi ies associa ed wi h he di e en s and ypes based on
hei successional s age. Fo his pu pose, when he leng h o he
ex ac ed g adien was <3 SD uni s, we used edundancy analysis (RDA)
(Te B aak, 1986) o assess he co ela ion be ween en i onmen al
a iables and he ungal composi ion. The o dina ion was based on he
abundance o each ungal g oup in each sample using CANOCO e sion
5.0 (Smilaue and Lepˇ
s, 2014). The o wa d selec ion was used o selec
signi ican explana o y a iables and only hose signi ican a he p <
0.05 le el we e included in he models. Bon e oni co ec ion was also
used a e including each en i onmen al a iable o be mo e es ic i e
in he selec ion o a iables. The s a is ical signi icance o en i on-
men al a iables was calcula ed using he Mon e Ca lo pe mu a ion es
(499 pe mu a ions). The e ec o s and age on phyla and EcM species
wi hin communi ies, including he e ec o he blocks wi hin each si e,
we e analyzed using a Pe MANOVA based on 999 pe mu a ions using
he adonis unc ion in he egan package. A simila i y pe cen ages
(SIMPER) ou ine was un o iden i y he ungal species esponsible o
he dissimila i y in he communi y s uc u es (Pa a icini e al., 2010).
The analysis was pe o med using PAST so wa e (Hamme e al., 2001).
In addi ion, we used Man el es s based on Pea son’s co ela ion o
de e mine he in luence o clima e and soil a iables on he soil ungal
communi y using B ay–Cu is dis ance o he a e ied OTU o al ma ix
and Euclidean dis ance o he scaled en i onmen al pa ame e s. Da a
we e scaled using R (R Co e Team, 2019) when needed o no malize
da a o analysis. Signi ican e ec s o he mo ypes and s and age we e
speci ically es ed by Linea Mixed E ec s (LME) models (Pinhei o e al.,
2016) wi h a nes ed design, whe e blocks we e nes ed in si e and
included as a andom ac o . S and age and he mo ype we e de ined as
ixed ac o s. LME models we e used o p e en alse-posi i e associa-
ions due o he ela edness s uc u e in he sampling. When signi ican
e ec s we e ound, we pe o med Tukey pos hoc speci ic con as s o
s and ages as jus wo he mo ypes we e s udied. We de e mined any
p e e ences o indi idual EcM ungal OTUs o a speci ic s and ype
using indica o species analyses (Du ˆ
en, 1997). This analysis was pe -
o med using he in e species package (C´
ace es and Legend e, 2009).
3. Resul s
3.1. Sequencing ou pu and ungal communi y composi ion
A o al o 2,258,090 sequencing eads, wi h a minimum o 35,404
eads pe sample, passed quali y il e ing, ep esen ing 2057 ungal
OTUs and 12 ungal phyla (Fig. 2). Ascomyco a and Basidiomyco a we e
he dominan phyla in all sample plo s, accoun ing o 90% o sequences.
Among he guilds, EcM ungi (44.1%) and sap o ophs (10%) we e he
mos dominan . O he g oups, including a buscula myco hizal ungi,
pa asi ic ungi, and animal pa hogens, ep esen ed <1% o sequences.
Howe e , we we e unable o assign a guild o 37.14% (N =765) o
OTUs. The axonomic dis ibu ion o all ungi ecognized a he phylum
le el and o guild g oups is shown in Fig. 2.
We ound signi ican di e ences in he dis ibu ion o Basidiomyco a
phyla g oups be ween he wo clima e ypes (p <0.05; Table 2). Fo
Basidiomyco a signi ican ly highe abundance alues we e ob ained in
plo s wi h a Sup aMedi e anean clima e ype. Howe e , only he
abundance o he Muco omyco a phyla was a ec ed by he s and age in
his s udy (Table 2).
3.2. Fungal ichness and di e si y acco ding o s and age and clima e
The o al ungi Shannon di e si y index (H) was signi ican ly
a ec ed by clima e (F =4.06, p =0.049) and by s and age (F =3.05, p =
0.050). Highe H alues we e ob ained o ungal communi ies in plo s
wi h a MesoMedi e anean clima e ype and in plo s wi h ma u e s ands
(Fig. 3). Howe e , no di e ences we e ound when compa ing he o al
ungal ichness alues o plo s wi h di e en clima e ypes o s and ages
(Fig. 3).
Analysis o speci ic guilds e ealed ha none o he guilds was
signi ican ly a ec ed by clima e ype (p >0.05; Table 3). Howe e ,
s and age had a signi ican e ec on EcM ungi (p <0.0001; Table 3),
wi h highe ichness alues ob ained o he la e-s age successional
s ands (p <0.05; Fig. 4). The ichness o EcM sho - and long-explo a ion
ypes inc eased wi h s and age (p <0.05). The e was no change in EcM
explo a ion ypes o o sap o oph ichness unde di e en clima e ypes
(p >0.05; Fig. 4).
3.3. En i onmen al ac o s a ec ing ungal composi ion
The CCA o phylum-le el axa ollowed by pe MANOVA analyses
con i med ha soil ungal communi ies a he ou s udied si es di e ed
(F =6.06, R2 =0.29, p =0.001, Fig. 5). The SIMPER analysis also
iden i ied ungal OTUs a he phylum le el ha we e esponsible o he
di e ences be ween he si es. Muco omyco a and Ascomyco a we e he
dominan phyla and we e gene ally in luen ial ac oss he pai wise
compa isons (Table S1). The cumula i e con ibu ion o he mos
in luen ial phyla o he dissimila i y be ween he s udied si es is p o-
ided (Table S1).
The clima ic and edaphic pa ame e s we e also co ela ed wi h soil
ungal communi y composi ion (p <0.05; Fig. 5). A Man el es
con i med ha clima ic a iables agg ega ely had a s onge e ec on
ungal communi y s uc u e ( =0.3076, p =0.001) han he g ouped
edaphic a iables ( =0.07184, p =0.012). Speci ically, he mean
annual empe a u e (Temp), mean annual p ecipi a ion (P ec), pH, and
phospho us (P) showed a highly signi ican in luence on he composi-
ion o he ungal communi y a he phylum le el (Fig. 5; Table 4). Only
clima ic pa ame e s (i.e., empe a u e and p ecipi a ion) in luenced
o al ungal guild composi ion (Table 4).
RDA o he o al EcM OTUs ollowed by pe MANOVA analyses
con i med ha EcM communi ies di e ed among he s ands (F =3.39,
R2 =0.13, p =0.001, Fig. 6A). The SIMPER analysis also iden i ied EcM
gene a esponsible o he di e ence be ween he s ands. Among he
di e en gene a, Co ina ius, Russula, Hebeloma, and Amani a we e
iden i ied as he dominan gene a ha we e gene ally in luen ial ac oss
he pai wise compa isons. The cumula i e con ibu ion o he mos
in luen ial EcM gene a o he dissimila i y be ween s ands is indica ed
in Table S2. Explana o y a iables such as s and age, clima e a iables,
and edaphic pa ame e s had a signi ican in luence on EcM ungal
communi y composi ion (p <0.05; Fig. 6A). The Man el es con i med
ha clima ic a iables (p ecipi a ion and empe a u e) agg ega ely had
a highe e ec on EcM ungal communi y s uc u e ( =0.4817, p =
0.001) han he g ouped edaphic a iables (pH, N, C, and P) ( =0.3786,
p =0.001).
Simila ly, RDA ollowed by pe MANOVA o EcM OTUs acco ding o
mycelial explo a ion ype also showed ha EcM explo a ion ypes
di e ed among he s udied s ands (F =6.52, R2 =0.23, p =0.001,
Fig. 6B). The SIMPER analysis also iden i ied he EcM explo a ion ypes
esponsible o he di e ence be ween he s ands: he sho -dis ance
explo a ion ype made he g ea es con ibu ion o dissimila i y ac oss
all s ands (Table S3). S and age ( =0.27, p =0.004), N ( =0.096, p =
0.038), and C ( =0.4, p =0.002) con ibu ed signi ican ly o he
a ia ion in EcM explo a ion ypes (Fig. 6B) among s ands. Mo eo e ,
aluable edible ungal species we e dis ibu ed di e en ly among he
hos s and ages. The gene a Bole us, T icholoma, and Lac a ius we e
associa ed wi h he la e o middle successional s ages, whe eas Amani a
was associa ed wi h he ea ly successional s age (Fig. 6A).
A o al o 49 EcM gene a we e associa ed wi h C. ladani e s ands.
Howe e , we ound indica o EcM gene a only in he ma u e and young
P. Ma ín-Pin o e al.
Fo es Ecology and Managemen 519 (2022) 120340
5
Fig. 2. K ona cha s showing (A) axonomic dis ibu ion o all ungi and (B) ungal guilds based on OTU ichness.
P. Ma ín-Pin o e al.

Fo es Ecology and Managemen 519 (2022) 120340
6
s ands in ou s udy a ea (Table S4). Among he o al EcM gene a, only
h ee (Inocybe, T icholoma, and Thelepho a) we e signi ican indica o
species (p < 0.05) o ma u e C. ladani e s ands, while ou EcM gene a
(Amani a, Hebeloma, Scle ode ma, and Pisoli hus) we e indica o species
associa ed wi h ea ly successional s ands. When conside ing he clima e
egion, he gene a Pisoli hus, Te ezia, and Hys e angium we e signi i-
can ly (p < 0.05) associa ed wi h he MesoMedi e anean en i onmen
ype, whe eas Bole us, Lac a ius, and Lacca ia we e associa ed wi h he
Sup aMedi e anean en i onmen (Table S4).
Finally, indica o species analysis o edible EcM a he species le el
Table 2
In luence o clima e o s and age on he abundance o speci ic ungal phyla.
Values in bold indica e signi ican e ec s (p <0.05).
Phylum Clima e S and age
F p- alue F p- alue
Ascomyco a 1.01 0.42 1.79 0.18
Basidiobolomyco a 0.96 0.33 1.89 0.16
Basidiomyco a 4.87 0.03 0.04 0.96
Calca ispo iellomyco a 1.51 0.34 0.26 0.76
Chy idiomyco a 0.000 0.97 1.09 0.34
En o hizomyco a 0.96 0.43 1.29 0.28
Kickxellomyco a 0.16 0.72 2.29 0.11
Mo ie ellomyco a 0.13 0.75 2.59 0.08
Muco omyco a 0.66 0.50 4.13 0.02
Olpidiomyco a 2.03 0.17 0.72 0.49
Rozellomyco a 2.54 0.24 1.50 0.23
Zoopagomyco a 0.19 0.65 1.29 0.28
Fig. 3. Shannon H index and ichness o he o al
soil ungal communi y de ec ed in plo s wi h a
Sup aMedi e anean (Sup) clima e ype o a Meso-
Medi e anean (Mes) clima e ype (A) o in young,
middle- (Mid) o la e-s age (Ma ) s ands (B). Means
we e compa ed using Tukey’s HSD es s, wi h
di e en lowe case le e s deno ing signi ican di -
e ences be ween means (p ≤0.05). The e o ba
shown o each mean alue is he s anda d de ia ion
o he mean. The absence o le e s abo e he ich-
ness mean alues indica es ha he e is no signi i-
can di e ence be ween means.
Table 3
In luence o clima e o s and age on he ichness o speci ic ungal guilds. Values
in bold indica e signi ican e ec s (p <0.05).
Guilds Clima e S and age
F p- alue F p- alue
Animal pa hogens 6.79 0.12 0.17 0.84
A buscula myco hizae 6.13 0.13 1.59 0.22
Bio oph gene alis s 0.46 0.56 0.19 0.82
Ec omyco hizal ungi 0.87 0.44 11.88 0.00
Fungal pa asi es 0.43 0.57 1.30 0.28
Lichenized ungi 0.99 0.33 1.72 0.19
Li e decompose s 0.99 0.42 0.95 0.39
Plan pa hogens 2.18 0.14 0.52 0.59
Sap o oph gene alis s 0.53 0.59 0.03 0.86
P. Ma ín-Pin o e al.
Fo es Ecology and Managemen 519 (2022) 120340
7
e ealed ha none o he indica o species we e signi ican ly associa ed
wi h s and age. Howe e , we ound ha Te ezia pseudolep ode ma was
signi ican ly associa ed wi h he MesoMedi e anean he mo ype (p =
0.006), while Co ina ius pu pu ascens (p =0.001) and Bole us edulis (p =
0.005) we e indica o species o he Sup aMedi e anean he mo ype.
These species showed a leas 98% simila i y o he e e ence sequences
(Table S5).
4. Discussion
In his s udy, ou ini ial hypo hesis ha he o al soil ungal di e si y
and ichness would be a ec ed by he successional s age o he Cis us
ladani e s ands a e he i e, was suppo ed by ou indings, assuming
ha he ungal species keep colonizing he habi a , esul ing in a cu-
mula i e inc ease in ichness in s ands a la e-s age succession (Cas a˜
no
e al., 2019). Ou indings di e ed om hose epo ed o a ecen
ch onosequence s udy in a Eu opean empe a e o es ha co e ed 137
yea s, which ound no ela ionship be ween o al ungal di e si y,
Fig. 4. Richness alues o EcM mycelial explo a ion
ypes and sap o ophic ungi de ec ed in plo s wi h a
Sup aMedi e anean (Sup) clima e ype o a Meso-
Medi e anean (Mes) clima e ype and in ea ly-
(Young), middle- (Mid), o la e-s age (Ma ) s ands.
EcM mycelial explo a ion ypes: EcM sho , sho -
dis ance explo a ion ype; EcM Long, long-dis ance
explo a ion ype; and EcM Mixed, mixed-dis ance
explo a ion ype. Means we e compa ed using
Tukey’s HSD es s, wi h di e en lowe case le e s
deno ing signi ican di e ences be ween means (p
≤0.05) o each g ouped se o da a acco ding o
guild ype and clima e/s and age. The e o ba
shown o each mean alue is he s anda d de ia ion
o he mean. The absence o le e s abo e mean
alues indica es ha he e is no signi ican di e -
ence be ween means.
Fig. 5. Canonical co espondence analysis o phyla de ec ed in Cis us ladani e s ands a ou si es wi h ei he a Sup aMedi e anean (SUP) clima e ype o a
MesoMedi e anean (ME) clima e ype. P ecipi a ion (P ec), empe a u e (Temp), pH, and phospho us (P) a e cons ained signi ican pa ame e s.
P. Ma ín-Pin o e al.
Fo es Ecology and Managemen 519 (2022) 120340
8
ichness, and s and age (Od iozola e al., 2020). In ou s udy, he e ec
o s and age on he ungal communi y could be due o he s ong
dominance o EcM ungi. As expec ed, we ound a g ea e p esence o
myco hizal ungi in la e-s age s ands han in he younge s ands:
49.38% o he o al OTUs ound in la e-s age s ands we e EcM species.
This also suppo s ou hypo hesis ha he e ec o s and age on ungal
communi ies would be mo e p onounced among myco hizal ungi,
which may be ela ed o changes in soil e ili y and inc easing ee oo
co e as s ands age (Cas a˜
no e al., 2019). Thus, he g ea e dependency
o ees in la e-s age s ands on hei ungal symbion s could compensa e
o he dec ease in soil e ili y and inc ease in plan nu ien s ess o e
ime (Cas a˜
no e al., 2019; Read and Pe ez-Mo eno, 2003). Unde such
condi ions, EcM ungi could also play a ole in p o ec ing hei hos s
om bio ic and abio ic s esses (Age e , 2001; De enne e al., 2019). In
addi ion, he e ec o s and age was obse ed o bo h sho -dis ance
and long-dis ance explo a ion- ypes o EcM species. Pe haps in Cis us-
domina ed sc ubland sys ems, whe e oo sys ems a e e y dense, his
ecological concep may be less ele an han in woodland sys ems,
whe e he low oo densi y o ma u e s ands leads o he g ea e domi-
nance o he long-dis ance explo a ion- ype o EcM ungi (Geml, 2019),
which he e o e p o ide a g ea e bene i o he ecosys em han he
sho -dis ance explo a ion ype.
Ou soil analyses also showed ha he ichness o sap o ophic ungi
did no change wi h s and age. Communi ies o sap o ophic ungi a e
known o be less in luenced by s and age han by subs a e ype, which is
p ima ily in luenced by he plan communi y (Gebaue and Taylo ,
1999) and, he e o e, could be in luenced by abio ic ac o s (Zaka ia and
Boddy, 2002). Consequen ly, sap ophy ic species end o be mo e s able
o e successional s ages and a e mo e in luenced by empe a u e o soil
humidi y (Sysouphan hong e al., 2010). In hese Cis us-domina ed
sc ubland ecosys ems, sap ophy ic ungi ound sui able niches
h oughou he successional cycle. In he ea ly s ages o succession, he
absence o ege a ion a ec s he abundance o plan -dependen
myco hizal ungi. Du ing his ime, sap ophy ic ungi ake ad an age
o he lack o compe i ion o sp ead apidly (He n´
andez-Rod íguez e al.,
2013). In he Medi e anean ecosys em, o ganic ma e apidly accu-
mula es on he soil su ace and, due o he lack o o e lap be ween peak
humidi y and empe a u e, mine aliza ion is ex emely slow (Ga cía and
He n´
andez, 1996; Rodeghie o e al., 2011). In addi ion, he high plan
densi y o Cis us-domina ed ecosys ems p o ec s he soil om di ec
sunligh , which gene a es adequa e humidi y o he de elopmen o
hese sap ophy ic ungi h oughou he en i e cycle o hese e y
pa icula sys ems (Media illa e al., 2021).
Ou indings a e in ag eemen wi h hose epo ed by p e ious
s udies o a ious biomes ha highligh ed he ole o clima ic and
edaphic a iables in o al ungal communi y composi ion (Canini e al.,
2020; Tede soo e al., 2014; Tede soo e al., 2021; Vˇ
e o ský e al.,
2020). Al hough in his s udy we ound clima e ypes a ec ed he
Basidiomyco a ichness, he gene al phyla composi ion we e mode a ely
associa ed clima e a iables such as empe a u e and ain all, as e-
po ed by Tede soo e al. (2014) and Newsham e al. (2016). This is
because highe ai empe a u e and soil mois u e le els enhance ungal
ac i i ies in he soil, enabling hem o swi ch om su i al o g ow h
s a egies (Newsham e al., 2016). Fu he mo e, edaphic a iables, such
as pH and phospho us, also a ec ungal communi ies, as epo ed by
Laube e al. (2008) and Nilsson e al. (2007). In his s udy, we also
obse ed a ela ionship be ween pH and EcM ungi a he han o he
guilds such as decompose s o pa hogenic ungi. This inding is in
ag eemen wi h p e ious s udies ha indica ed a di ec ela ionship
be ween pH and EcM ungi (Canini e al., 2019) and a nega i e ela-
ionship wi h o he unc ional g oups such as sap o ophs, plan and
animal pa hogens, and mycopa asi ic ungi (Canini e al., 2019). This is
because EcM species can play an impo an ole in plan g ow h in acidic
soils, such as Cis us-domina ed sc ublands, whe e he a ailabili y o
essen ial nu ien s is low (Mengel and Ki kby, 2001). Fu he mo e,
Table 4
Signi icance o explana o y a iables om cons ained mul i a ia e analysis,
CCA o phyla, and all ca ego ized guilds. The Hellinge - ans o med ungal
communi y was conside ed. Values in bold indica e signi ican e ec s (p <0.05).
Va iable Explains
%
Con ibu ion
%
pseudo-
F
p-
alue
Phyla pH 9.3 32.0 4.6 0.014
Tempe a u e 6.1 20.8 3.1 0.014
Phospho us 3.9 13.5 2.1 0.002
P ecipi a ion 4.7 16.1 2.6 0.042
Guilds Tempe a u e 11.6 35.8 5.9 0.002
P ecipi a ion 11.1 28.7 5.6 0.010
Fig. 6. Redundancy analysis o EcM gene a (A) and acco ding o EcM explo a ion ype (B) ela ed o explana o y clima ic and edaphic a iables g ouped by s and
ype. Isolines ep esen p ecipi a ion using a gene alized addi i e model- i ing me hod (F =27.0, p <0.000). EcM explo a ion ype: C_SD_MDS, con ac /sho -
dis ance/medium-dis ance smoo h wi h hyd ophilic hyphae; MDM_MDF, medium-dis ance ma /medium-dis ance inge wi h hyd ophobic hyphae.
P. Ma ín-Pin o e al.
Fo es Ecology and Managemen 519 (2022) 120340
9
phospho us up ake om he soil is acili a ed indi ec ly ia myco hizal
symbiosis, whe e he hos plan ob ains phospho us p ima ily om he
ungal pa ne , while he ungus bene i s om plan -de i ed educed
ca bon (Joh i e al., 2015). Simila ly, ou inding ha empe a u e and
p ecipi a ion explain some o he a ia ions in EcM assemblages was
simila o he indings epo ed by Pena e al. (2017). Howe e , he
agg ega e e ec o clima e on he s uc u e o he EcM ungal commu-
ni y in his s udy could also be ela ed o si e e ec s, such as he poo soil
condi ions and ecu en i es, ha assis ed he co-e olu ion o he hos
species (C. ladani e ) and ungal symbion s (De enne e al., 2019) in he
s udy a ea.
The indica o species analysis e ealed ha EcM ungal gene a ha e
a p e e ence o a pa icula s and successional s age, sugges ing ha
s and succession can in luence he s uc u e o he EcM ungal com-
muni y in a Cis us-domina ed ecosys em. Fo example, species belonging
o he gene a Amani a, Hebeloma, Scle ode ma, and Pisoli hus we e
associa ed wi h ea ly successional s ands. Al hough hese gene a a e
cha ac e is ic o la e-s age species in Pinus s ands (Chu-Chou and G ace,
1982; Visse , 1995), when hese species a e associa ed wi h Cis us spp.,
hey beha e as ea ly-s age myco hizal species. This suppo s indings
epo ed by He n´
andez-Rod íguez e al. (2013) who ound spo oca ps o
hese species when C. ladani e was a he ea ly s ages o de elopmen .
Simila ly, we ound ha Inocybe, T icholoma, and Thelepho a we e
associa ed wi h la e-s age s ands. Howe e , he p esence o mos o hese
species has been epo ed a bo h he ea ly and la e s ages o C. ladani e
de elopmen (He n´
andez-Rod íguez e al., 2013), indica ing ha hese
gene a a e also mul i-s age species ha migh be able o ui unde
di e en ecological condi ions and ha hey ha e wide subs a e e-
qui emen s. Fu he mo e, he ne wo k o myco hizal mycelium could
also play a ole in educing e osion ha could po en ially occu when he
i s ain alls a e a i e. Thus, he conse a ion and ehabili a ion alue
o Cis us sc ubland, which is ich in a ange o aluable species, along
wi h he de elopmen o he s ands, is conside able due o he sho li e
cycle (18–20 yea s) o C. ladani e . Howe e , when s ands o his species
wi he and die, hey c ea e a e y high isk o o es i es (O ia De Rueda
e al., 2008). Thus, he managemen o Cis us sc ublands should in ol e
measu es o p e en uel con inui y in he sys em. In his sense, mosaic
s and s uc u e managemen , including young, in e media e, and
ma u e C. ladani e s ands simul aneously, may help o conse e ungal
ichness and di e si y and acili a e he p oduc ion o some highly
p ized and so a e edible species, while p e en ing he occu ence o
wild i es in hese a eas by educing uel loads.
In he absence o i e, Cis us-domina ed sc ublands will be p og es-
si ely occupied by o he species such as Que cus ilex L. o Que cus py -
enaica Willd. (He n´
andez-Rod íguez e al., 2013). Because o his,
conside a ion o i e-p e en ion ea men s is also i al as Cis us-domi-
na ed ecosys ems can ac as a b idge, p o iding myco hizal inoculum
ha can colonize ee oo s as a new s and de elops o in ec ing new
oo s in younge s ands du ing he eco e y o o es s ands a e a i e,
he eby, enabling he sys em o p oduce g ea e quan i ies o myco -
hizal edible spo oca ps. In addi ion, he dispe sion and sha ing o
myco hizal species om nea by hos o es s, such as Que cus s ands,
wi h Cis us-domina ed s ands, o ice e sa, should be conside ed in he
managemen s a egy o a Cis us-domina ed sys em as he p esence o
such o es s, which ac as al e na i e hos s o ese oi species, enables a
wide di e si y o ungal species o dispe se o o o pe sis in his
ecosys em (Tomao e al., 2017). The e o e, a e a i e, manage s o
hese ecosys ems can choose o keep sc ublands, o main ain he ungal
ichness and di e si y associa ed wi h i , o o use C. ladani e as a ool o
p o ide ungal p opagules o he es ablishmen o a new o es s and in
he a ea (He n´
andez-Rod íguez e al., 2015b).
5. Conclusions
We obse ed a s ong in luence o s and age on he gene al di e si y
indices and on he ichness o he s udied ungal communi y, including
he EcM ungal communi y. The ungal communi y composi ion was
p ima ily d i en by clima ic and edaphic ac o s, whe eas s and age only
had a weak in luence on he ungal communi y. Analysis a he phylum
le el e ealed ha edaphic a iables such as phospho us and pH
signi ican ly in luenced he composi ion o he ungal communi y.
O dina ion analysis o EcM gene a showed ha s and age and clima e
ype had a signi ican d i ing e ec on his communi y. Analysis o he
EcM explo a ion ype also e ealed an e ec o s and age, wi h a g ea e
ichness o sho -dis ance explo a ion- ype axa in he oldes s ands.
Finally, some gene a, such as Inocybe, T icholoma, and Thelepho a, we e
signi ican ly associa ed wi h ma u e s ands, whe eas Amani a, Hebeloma,
Scle ode ma, and Pisoli hus we e associa ed wi h ea ly successional
s ands. Fu he mo e, he gene a Pisoli hus, Te ezia, and Hys e angium
we e signi ican ly associa ed wi h he MesoMedi e anean en i onmen
ype whe eas Bole us, Lac a ius, and Lacca ia we e associa ed wi h he
Sup aMedi e anean en i onmen . Rema kably, C. ladani e -domina ed
sc ublands suppo di e se ungi, including highly ega ded edible
species, such as Bole us and Lac a ius, unde speci ic clima e, soil, and
s and ac o condi ions. These indings sugges ha C. ladani e sc ub-
lands may play an impo an ole in he eco e y o o es s ands a e a
i e by p o iding myco hizal inoculum ha can colonize ee oo s as
new s ands de elop. Al hough managemen canno modi y clima e pa-
ame e s such as p ecipi a ion and empe a u e, managemen s a egies
ha conside mosaic landscapes o educe he se e i y o po en ial i es
in hese widely dis ibu ed sc ubland ecosys ems could p o ide sui able
habi a s o p omo ing ungal di e si y, p oduc ion, and unc ion.
Fu he mo e, he e en ion o la e-successional s ands as pa o a mosaic
landscape managemen app oach would ha e impo an implica ions
o sc ubland loo mic ohabi a s, such as soil e ili y imp o emen ,
which a e impo an o mac o ungal occu ence and p oduc ion in
Cis us-domina ed sc ublands. Ou indings could se e o guide u u e
s udies in many o he coun ies wi h simila con ex s o op imize Cis us-
domina ed ecosys ems ha a e a ec ed by ecu en i es.
CRediT au ho ship con ibu ion s a emen
Pablo Ma ín-Pin o: Concep ualiza ion, Me hodology, In es iga-
ion, Supe ision, W i ing – e iew & edi ing. Juan And ´
es O ia-de-
Rueda: Supe ision, Me hodology. Ta ek Dejene: In es iga ion,
Me hodology, W i ing – e iew & edi ing. Olaya Media illa: In es i-
ga ion, Me hodology. Ma ía He n´
andez-Rod íguez: In es iga ion,
Me hodology. Jos´
e A. Reque: Supe ision. Ignacio Sanz-Beni o:
W i ing – e iew & edi ing. Ma ía San os: In es iga ion, Me hodology.
J´
ozse Geml: Concep ualiza ion, Me hodology, In es iga ion, Supe i-
sion, W i ing – e iew & edi ing.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgmen s
This esea ch wo k was pa ially suppo ed by he MYCOINFOR
(PID2019-105188RB-I00), which is unded by he Spanish Minis y o
Science and Inno a ion. We a e g a e ul o e e yone ha was in ol ed
in he ieldwo k. We would like also hank o Luis San os o his suppo
in s a is ical e iew.
Appendix A. Supplemen a y ma e ial
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. o eco.2022.120340.
P. Ma ín-Pin o e al.