Influence of stand age and site conditions on ectomycorrhizal fungal dynamics in Cistus ladanifer-dominated scrubland ecosystems

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.