Dieback and Replacement of Riparian Trees May Impact Stream Ecosystem Functioning

Vol.:(0123456789)
Mic obial Ecology (2024) 87:32
h ps://doi.o g/10.1007/s00248-024-02343-w
RESEARCH
Dieback andReplacemen o Ripa ian T ees May Impac S eam
Ecosys em Func ioning
Albe oAlonso1 · LuzBoye o1,2 · Alejand oSolla3 · Ve ónicaFe ei a4
Recei ed: 27 Sep embe 2023 / Accep ed: 3 Janua y 2024
© The Au ho (s) 2024
Abs ac
Alde s a e ni ogen (N)- ixing ipa ian ees ha p omo e lea li e decomposi ion in s eams h ough hei high-nu ien
lea li e inpu s. While alde s a e widesp ead ac oss Eu ope, hei popula ions a e a isk due o in ec ion by he oomy-
ce e Phy oph ho a ×alni, which causes alde dieback. Mo eo e , alde dea h opens a space o he es ablishmen o an
agg essi e N- ixing in asi e species, he black locus (Robinia pseudoacacia). Shi s om ipa ian ege a ion con aining
heal hy o in ec ed alde and, e en ually, alde loss and eplacemen wi h black locus may al e he key p ocess o lea li e
decomposi ion and associa ed mic obial decompose assemblages. We examined his ques ion in a mic ocosm expe imen
compa ing h ee ypes o lea li e mix u es: one ep esen ing an o iginal ipa ian o es composed o heal hy alde (Alnus
lusi anica), ash (F axinus angus i olia), and popla (Populus nig a); one wi h he same species composi ion whe e alde had
been in ec ed by P. ×alni; and one whe e alde had been eplaced wi h black locus . The expe imen las ed six weeks, and
e e y wo weeks, mic obially d i en decomposi ion, ungal biomass, ep oduc ion, and assemblage s uc u e we e measu ed.
Decomposi ion was highes in mix u es wi h in ec ed alde and lowes in mix u es wi h black locus , e lec ing di e ences
in lea nu ien concen a ions. Mix u es wi h alde showed dis inc ungal assemblages and highe spo ula ion a es han
mix u es wi h black locus . Ou esul s indica e ha alde loss and i s eplacemen wi h black locus may al e key s eam
ecosys em p ocesses and assemblages, wi h impo an changes al eady occu ing du ing alde in ec ion. This highligh s he
impo ance o main aining hea hy ipa ian o es s o p ese e p ope s eam ecosys em unc ioning.
Keywo ds Alde · Alnus lusi anica· Black locus · Robinia pseudoacacia· Phy oph ho a ×alni· Lea li e decomposi ion
In oduc ion
The unc ioning o s eam ecosys ems can be highly in lu-
enced by changes in he ipa ian o es [1, 2]. This applies
especially o headwa e s eams, whe e he majo basal
esou ce is he alloch honous o ganic ma e ial p o ided
by he ipa ian ege a ion [3], mainly in he o m o lea
li e [4–6]. Gi en ha di e en ee species p oduce
lea li e wi h di e en ai s [7–9], species composi ion
o he ipa ian o es can de e mine how his lea li e
is p ocessed in he s eam and he e o e pa ially egu-
la e i s decomposi ion a es, he ans e o ene gy and
nu ien s be ween ecosys em compa men s [10], and he
cha ac e is ics o s eam assemblages in ol ed in hese
p ocesses [11–13]. In consequence, changes in ipa ian
species composi ion, which can occu h ough species loss
(e.g.,caused by pa hogenic diseases o he long e m esul
o biological in asions [4, 14]), species gain o species
eplacemen (e.g., a ini ial s ages o biological in asions
* Albe o Alonso
albe [email p o ec ed]
1 Depa men o Plan Biology andEcology, Facul y
o Science andTechnology, Uni e si y o  heBasque
Coun y (UPV/EHU), Leioa, Spain
2 Basque Founda ion o Science, IKERBASQUE, Bilbao,
Spain
3 Facul y o Fo es y, Ins i u e o Dehesa Resea ch
(INDEHESA), Uni e sidad de Ex emadu a, A enida Vi gen
del Pue o 2, 10600Plasencia, Spain
4 Ma ine andEn i onmen al Sciences Cen e (MARE),
Aqua ic Resea ch Ne wo k (ARNET), Depa men o Li e
Sciences, Uni e si y o Coimb a, Calçada Ma im de F ei as,
3000-456Coimb a, Po ugal
A.Alonso e al. 32 Page 2 o 11
[15]), a e expec ed o be highly ele an o p edic changes
in s eam ecosys em unc ioning.
Alde s (Alnus spp.) a e key ipa ian ees ac oss Eu ope
and o en he only ni ogen (N)- ixing na i e ee species
p esen [16]. Thei lea es a e so and high in N concen a-
ion [16], which makes hem highly a ac i e o mic obial
decompose s and de i i o es, hence allowing apid lea
decomposi ion in s eams [17]. Howe e , alde s a e su e -
ing widesp ead mo ali y due o he Alnus-speci ic oomy-
ce especies complex Phy oph ho a alni [18–21]. In Spain
and Po ugal, alde mo ali y has been obse ed since he
mid-2000s and P. ×alni (B asie & S.A. Ki k) Husson, Ioos
andMa çaiswas i s isola ed in 2009 and 2010, espec-
i ely [22, 23]. In he no he n egions, disease se e i y has
been high, leading o he disappea ance o mos o he ees
in As u ias and no he n Galicia. A he sou he n limi o
P. ×alni dis ibu ion, occu ing in Je e i e (Plasencia,
Spain) [24] and Cei a i e (A ouce, Po ugal) [25], dis-
ease se e i y is lowe han in he no h. T ee in ec ion is
p oduced by zoospo es in he oo s o in he unk du ing
loods, leading o oo o , colla o , small-size, spa se and
o en chlo o ic oliage, c own dieback, and ee mo ali y.
Mo ali y a es each almos 100%, wi h young ees usu-
ally dying in ew mon hs and old ees dying in yea s while
losing i ali y p og essi ely [18, 19]. Lea li e o Alnus
lusi anica Ví , Douda and Mandák in ec ed ees has highe
nu ien concen a ion han ha om heal hy ees due o
educed nu ien eso p ion be o e senescence, leading o
as decomposi ion o lea li e [24].
When key ee species disappea om o es s, exo ic spe-
cies can eadily occupy hei niche [26]. Ripa ian o es s a e
pa icula ly p one o in asions because s eams ac as co i-
do s a o ing p opagule anspo , cause na u al dis u bances
(i.e., loods) ha open canopy gaps, and al e mic o-clima ic
condi ions hus a o ing plan es ablishmen [27–29]. Occu -
ence o in asion is highe in ipa ian a eas a ec ed by
human ac i i ies (e.g., o es y, ag icul u e o u baniza ion),
whe e p opagule p essu e is highe and he coloniza ion o
shade sensi i e in asi e species is a o ed by la ge o es
gaps [15, 30]. A likely species o eplace alde a e i s dis-
appea ance is he black locus (Robinia pseudoacacia L.),
an N- ixing ee species na i e o No h Ame ica ha has
become a majo in asi e species in Eu opean ipa ian o es s
due o i s as g ow h, high esis ance o dis u bance, and
low nu ien equi emen s [26, 31, 32]. Despi e he high N
concen a ion o black locus lea li e , i decomposes mo e
slowly han lea li e o many na i e ee species (e.g., Salix
a ocine ea B o ., F axinus angus i olia Vahl. o Populus
alba L.), possibly because i s high concen a ion o second-
a y compounds and lignin nega i ely a ec s mic obial colo-
niza ion and mac oin e eb a e assemblages [33–35].
We explo ed whe he alde in ec ion by P. ×alni and
subsequen alde eplacemen by black locus a ec s s eam
ecosys em unc ioning. Mic obially-d i en lea li e
decomposi ion and he cha ac e is ics o associa ed ungal
assemblages we e assessed. Wi h his aim, we conduc ed a
mic ocosm expe imen using lea li e and simula ed he
ollowing scena ios: (1) a na i e ipa ian o es con ain-
ing ash (F. angus i olia), popla (Populus nig a L.), and
heal hy alde (A. lusi anica, p e iously Alnus glu inosa (L.)
Gae n.); (2) he same o es bu wi h alde in ec ed by P.
×alni, ep esen ing an ea ly s age o an epidemic; and (3) he
same o es bu wi h alde eplaced by black locus , ep e-
sen ing a pos -epidemic s age. To explo e di e ences among
na i e ash, na i e popla , heal hy and in ec ed na i e alde ,
and in asi e black locus , and acili a e he unde s anding
o in e ac ions in lea mix u es, including di e si y e ec s
on decomposi ion and ungal biomass, monocul u es o all
lea li e ypes we e also assessed. Based on he main ai s
o di e en lea li e ypes, we hypo hesized he ollowing:
(i) Scena io 2, wi h in ec ed alde , would show highe
lea li e decomposi ion a e, ungal biomass, and
ungalspo ula ion a e han scena io 1, wi h heal hy
alde , since lea li e o in ec ed alde is iche in
nu ien s and mo e labile [24], hus possibly enhanc-
ing decomposi ion and ungal ac i i y in mix u es
[12].
(ii) Scena io 3, wi h black locus , would show lowe
lea li e decomposi ion a e, ungal biomass, and
ungalspo ula ion a e han scena ios 1 and 2 (wi h
heal hy and in ec ed alde , espec i ely), because o
he highe lignin and polyphenol concen a ion in
black locus lea li e , which po en ially slows down
decomposi ion and ungal ac i i y in mix u es [12],
despi e bo h black locus and alde ha ing high con-
cen a ion o nu ien s [33, 34].
(iii) Fungal assemblages would be al e ed in scena ios 2
and 3 compa ed wi h scena io 1, due o s ong sub-
s a e p e e ences o ungal mic oo ganisms [11],
he change being highe unde scena io 3 han unde
scena io 2 because ungi would be mo e a ec ed by
species composi ion change [12] han by alde in ec-
ion [24].
Ma e ials andMe hods
Lea Li e Collec ion
The ou plan species used in he expe imen (ash,
popla , alde , and black locus ) a e b oadlea decidu-
ous ees ha ange widely in lea li e ai s (Table1).
Lea es o popla , ash and black locus we e collec ed
immedia ely a e na u al abscission om he loo in
he loodplain o he Mondego i e (Coimb a, cen al
Dieback andReplacemen o Ripa ian T ees May Impac S eam Ecosys em Func ioning Page 3 o 11 32
Po ugal), a Choupalinho (40°12′4.7″N, 8°25′42.9″W, in
au umn 2020), Pa que Ve de (40°12′3.2″N, 8°25′29.7″W,
in au umn 2022), and Ma a Na ional do Choupal
(40°13′4.3″N, 8°26′28.4″W, in au umn 2022), espec i ely.
Senescen alde lea es we e gen ly de ached om heal hy
ees (≤ 5% c own anspa ency es ima ed isually) and
ees in ec ed by P. ×alni (≥ 60% c own anspa ency)
loca ed in he loodplain o he Je e i e (Plasencia,
Spain; 40°1′51.2″N, 6°4′46.0″W, in au umn 2017) [24].
Isola ions o P. ×alni om ba k samples, including he
cambium [20], con i med in ec ion o he ees, whe eas
no pa hogen was isola ed om ba k samples o heal hy
ees. Lea li e was ai -d ied a oom empe a u e in he
labo a o y and s o ed in he da k un il needed.
Lea Li e Cha ac e iza ion
Ini ial cha ac e iza ion was pe o med using h ee epli-
ca es pe lea li e ype. Ai -d ied lea li e was milled
in o ine powde (< 0.5 mm; Re sch MM 400, Haan, Ge -
many), o en-d ied a 60°C o 48 h, and used o chemical
de e mina ions. Ca bon (C) and N concen a ions we e
assessed by iso ope a io mass spec opho ome y (IRMS
The mo Del a V ad an age wi h a Flash EA-1112 se ies;
The mo Fishe Scien i ic Inc., Wal ham, USA). Phospho-
ous (P) concen a ion was assessed by he asco bic acid
me hod a e basic diges ion wi h sodium pe sul a e and
sodium hyd oxide [36]. To al polyphenol concen a ion
was ob ained by he Folin-Ciocal eu me hod [36] and
lignin concen a ion by he Goe ing- an Soes me hod
[37]. Concen a ions we e exp essed as % d y mass (DM).
Ini ial li e oughness (kPa) was es ima ed wi h a pene-
ome e ( oddiame e 1.55 mm) a e one hou o soaking
in dis illed wa e [36].
Expe imen al P ocedu e
The expe imen al design included eigh ea men s: i e
monocul u es (ash, popla , heal hy and in ec ed alde , and
black locus ) and h ee mix u es, called scena ios, wi h h ee
species each: ash, popla , and heal hy alde (scena io 1);
ash, popla , and in ec ed alde (scena io 2); and ash, popla ,
and black locus (scena io 3). Each ea men included h ee
eplica es collec ed a each o ou sampling da es; hus, he
expe imen comp ised 96 mic ocosms in o al. Be o e he
beginning o he expe imen , lea li e was mois ened wi h
dis illed wa e , and 6-mm diame e discs we e cu a oiding
cen al eins (excep o he na ow ash lea es, whe e discs
included he ein in he cen e ). Discs we e ai -d ied a oom
empe a u e o 72 h and weighed (± 0.1 mg) in g oups o
12, ei he o he same species (monocul u e) o 4 discs pe
species (mix u es, wi h each species weighed indi idually).
Discs we e hen dis ibu ed in 100-mL E lenmeye lasks
(mic ocosms), which we e assembled on an o bi al shake
(100 pm; GFL 3017, P o iLab24 GmbH, Be lin, Ge many)
and kep unde con olled condi ions (21°C and 12 h ligh :12
h da k pho ope iod).
Fo he i s se en days, mic ocosms we e supplied daily
wi h 40 mL o a mic obial inoculum <24 h old, o allow o
leaching and mic obial coloniza ion o lea li e discs. The
inoculum was p epa ed by incuba ing a di e se mix u e o
lea li e a di e en decomposi ion deg ees in a glass ja
wi h 4 L o il e ed (100 μm) s eam wa e and ae a ion, kep
a 21°C, wi h wa e enewal e e y 24 h. The li e and wa e
we e collec ed in Oc obe 2022 om Candal s eam (Lousã
moun ain, cen al Po ugal; 40°4′44.7″N, 8°12′11.3″W), an
oligo ophic s eam wi h ipa ian ege a ion a he sampling
si e domina ed by Eu opean ches nu ees (Cas anea sa i a
Mill.), and om whe e he ee species used in his s udy
we e absen [6]. A se o h ee mic ocosms pe ea men
Table 1 Ini ial ai s o he di e en lea li e ypes used in he s udy (mean ± s.e.; n=3): ca bon, ni ogen, phospho us, polyphenol, and lignin
concen a ions, elemen al mola a ios, and lea oughness
Fo each ai , di e en le e s indica e signi ican di e ences among lea li e ypes, analyzed wi h linea models
*p < 0.05; **p < 0.01; ***p < 0.001
Lea li e ai s F axinus angus i olia Populus nig a Alnus lusi anica heal hy Alnus lusi anica in ec ed Robinia pseudoacacia
Ca bon (C; % DM)*** 44.16 ± 0.10c42.00 ± 0.20e47.62 ± 0.09a46.48 ± 0.19b43.44 ± 0.21d
Ni ogen (N; % DM)*** 0.75 ± 0.02d0.87 ± 0.02c3.80 ± 0.73a2.52 ± 0.08a1.33 ± 0.01b
Phospho us (P; %
DM)***
0.17 ± 0.01a0.14 ± 0.01b0.06 ± 0.00cd 0.14 ± 0.05abc 0.04 ± 0.01d
Polyphenol (% DM)** 9.62 ± 0.28b16.30 ± 4.13ab 8.50 ± 0.83b9.83 ± 0.33b11.86 ± 0.43a
Lignin (% DM)*** 17.99 ± 0.35c35.88 ± 0.11a37.06 ± 1.99ab 36.74 ± 1.56ab 34.06 ±0.43b
C:N*** 59.25 ± 1.92a48.52 ± 1.30b13.69 ± 3.00d18.49 ± 0.53d32.70 ± 0.26c
C:P*** 253.69 ± 8.04c304.56 ± 14.39b845.74 ± 54.69a422.71 ± 114.52bc 1092.26 ± 165.87a
N:P*** 4.29 ± 0.22d6.28 ± 0.23c65.85 ± 9.35a23.22 ± 6.66b33.38 ± 5.00b
Toughness (kPa)*** 801 ± 96ab 898 ± 77a609 ± 52b551 ± 111abc 382 ± 9c
A.Alonso e al. 32 Page 4 o 11
(i.e., 24 mic ocosms) was sac i iced a e he condi ioning
pe iod (day 0) and p ocessed as he expe imen al mic o-
cosms (see below), o ob ain a co ec ion ac o o es ima e
ini ial, pos -leaching, li e DM.
A day 0, all o he expe imen al mic ocosms we e sup-
plied wi h 40 mL o il e ed s eam wa e (Candal s eam),
which was enewed e e y 3.5 days. Th ee eplica es o each
ea men we e sac i iced a days 14, 28, and 42 o assess
emaininglea li e mass, ungal biomass, and conidial p o-
duc ion. All li e discs we e ozen a −20°C, lyophilized
o e nigh (Lablyo Mini, F ozen in Time, No h Yo kshi e,
UK), and weighed o de e mina ion o DM emaining, wi h
species in mix u es weighed indi idually.
Fungal Conidial P oduc ion
A each sampling da e (days 14, 28, and 42), conidial sus-
pensions om he sac i iced mic ocosms (40 mL) we e
pou ed in o 50-mL cen i uge ubes, p ese ed wi h 2 mL o
37% o malin, adjus ed o a olume o 45 mL wi h dis illed
wa e , and s o ed in he da k un il p ocessed. Samples we e
p ocessed o de e mine spo ula ion a es and assemblage
s uc u e o aqua ic hyphomyce es, a polyphyle ic g oup o
aqua ic ungi assumed o be majo mic obial decompose s
[38, 39]. Each conidial suspension ecei ed 100 μL o 0.5
% T i on X-100 and was homogenized wi h a magne ic s i -
e . Then, 10 mL was il e ed h ough ni o-cellulose il e s
(25-mm diame e , 5-μm po e size; Sa o ius S edim Bio-
ech GmbH, Goe ingen, Ge many), and il e s we e s ained
wi h 0.05% co on blue in 60% lac ic acid. Conidia we e
iden i ied and coun ed wi h a mic oscope (Leica, DM1000,
We zla , Ge many) a ×200 magni ica ion [36]. Spo ula ion
a es we e exp essed as numbe o conidia mg−1 DM d−1,
and species ichness was exp essed as numbe o species
pe sample.
Fungal Biomass
A days 14 and 42, ou discs om each species pe mic o-
cosm (i.e., 4 discs in monocul u es o he 4 discs o each
species in mix u es) we e used o de e mine ungal biomass
om e gos e ol concen a ion [36]. Discs we e weighed o
de e mine DM and e gos e ol was ex ac ed in 10 mL o
alkaline me hanol (8 g KOH/L) in a ho ba h (80°C, 30 min),
pu i ied by solid phase ex ac ion (Wa e s Sep-Pak Vac RC,
500 mg, Tc18 ca idges; Wa e s Co p., Mil o d, MA, USA),
and quan i ied wi h high-pe o mance liquid ch oma og a-
phy (Dionex DX-120; Sunny ale, CA, USA) by measu ing
abso bance a 282 nm [36]. E gos e ol concen a ion was
con e ed in o ungal biomass assuming 5.5 μg e gos e ol
mg−1 ungal DM [40], and esul s we e exp essed as mg
ungal DM.
Da a Analyses
The ac ion o lea li e DM emaining pe species and
mix u e was calcula ed by di iding DM emaining by ini ial
DM. Decomposi ion a es (k, day−1) we e calcula ed o each
species and mix u e assuming an exponen ial decay model,
h ough linea eg ession o he ln- ans o med ac ion o
DM emaining o e ime, conside ing he in e cep ixed a
ln(1)=0. Ne di e si y, complemen a i y, and selec ion e ec s
on decomposi ion we e also calcula ed [41]; he ne di e si y
e ec was he di e ence be ween obse ed and expec edlea
li e mass loss (LML, calcula ed as he di e ence be ween
ini ial DM and DM emaining di ided by ini ial DM), wi h
expec ed ac ion LML calcula ed as he mean o monocul u e
alues aking in o accoun he p opo ion o each species in
he mix u e; he complemen a i y e ec was he a e age de i-
a ion om he expec ed ac ion LML in a mix u e mul iplied
by mean ac ion LML in monocul u es and he numbe o
species in he mix u e; and he selec ion e ec was he co a i-
ance be ween ac ion LML o species in monocul u e and he
a e age de ia ion om expec ed ac ion LML o species in
he mix u e, mul iplied by he numbe o species in he mix-
u e. Ne di e si y, complemen a i y, and selec ion e ec s on
ungal biomass was calcula ed in he same way using ungal
biomass o each species in monocul u es and mix u es.
Ini ial lea li e ai s we e compa ed among lea li e
ypes (ash, popla , heal hy alde , in ec ed alde , and black
locus ) wi h linea models and ANOVA (gls unc ion o he
“nlme” R package), wi h species as ixed ac o . E ec s o
lea li e ype ( i e ypes) and scena io (1, 2 o 3) on di e -
en esponse a iables we e analyzed sepa a ely. To assess
lea li e decomposi ion, we used analysis o co a iance
(ANCOVA, ao unc ion o he “s a s” R package), wi h
ac ion DM emaining as a dependen a iable, ea men
(lea li e ype o scena io) as a ca ego ical ac o and ime
as a co a ia e. To assess ungal biomass, spo ula ion a e
and species ichness, and ne , complemen a i y, and selec-
ion e ec s on decomposi ion and ungal biomass, we used
linea models and ANOVA, wi h ea men (lea li e ype
o scena io) and ime as ixed ac o s. Signi ican di e -
ences be ween ea men s (α = 0.05) we e analyzed wi h
Tukey’s es s, o Fishe ’s LSD es s when Tukey’s es s did
no iden i y di e ences among ea men s [i..e., o species
ichness and ne complemen a i y and selec ion e ec s; ghl
unc ion o he “mul comp” R package; [42]]. Di e ences
in ungal assemblages among ea men s and sampling da es
we e explo ed wi h non-me ic mul idimensional scaling
(NMDS) based on he B ay-Cu is simila i y index applied
o an abundance ma ix (me aMDS unc ion o he “ egan”
R package), ollowed by pe mu a ional mul i a ia e analysis
o a iance (PERMANOVA; adonis unc ion o he “ egan”
R package). An indica o alue index (mul ipa unc ion o
he “indicspecies” R package) was used o iden i y he mos
Dieback andReplacemen o Ripa ian T ees May Impac S eam Ecosys em Func ioning Page 5 o 11 32
ep esen a i e axa o each assemblage. No mal dis ibu ion
o esiduals was assessed by heShapi o-Wilk es , and da a
we e log- ans o med when non-no mal dis ibu ion was
de ec ed. All analyses we e pe o med in R so wa e [43].
Resul s
Lea Li e T ai s
Ini ial ai s a ied wi h he ype o lea li e (Table1, Table
S1). Na i e species (ash, popla , and heal hy alde ) showed
a g adien , wi h N and lignin concen a ions and C:P and
N:P a ios being highes in heal hy alde and lowes in ash,
while P concen a ion and C:N showed he opposi e pa e n;
C concen a ion was lowes in popla and highes in heal hy
alde . Heal hy and in ec ed alde di e ed in C concen a ion
and C:P and N:P a ios, which we e highe in heal hy alde .
Black locus had highe polyphenol concen a ion han he
o he species excep popla ; he lowes oughness, al hough
no signi ican ly di e en om in ec ed alde ; and highe
lignin concen a ion han ash and lowe han popla . Black
locus had lowe N concen a ion han alde , bu highe han
ash and popla ; he lowes P concen a ion, bu no signi i-
can ly di e en om heal hy alde ; and he second lowes C
concen a ion, a e popla (Table1).
Lea Li e Decomposi ion
Lea li e ypes signi ican ly di e ed in hei decomposi ion
a es (p- alue < 0.001; Table S2, Fig.1A), wi h (i) na i e spe-
cies showing a g adien om lowes in ash o highes in alde ;
(ii) in ec ed alde ending o decompose as e han heal hy
alde , wi h he di e ence being non-signi ican ; (iii) and
black locus decomposing mo e slowly han alde (Fig.1A).
Lea li e decomposi ion a ied wi h he scena io (p- alue =
0.008), being signi ican ly highe (56%) in scena io 2 han in
scena io 3, 23% highe in scena io 2 han in scena io 1, and
21% lowe in scena io 3 han in scena io 1, al hough he las
wo di e ences we e non-signi ican (Table S2, Fig.1B). All
scena ios had highe decomposi ion a es han expec ed om
monocul u es (19–43%), wi h a posi i e ne di e si y e ec in
all o hem (Fig.1B). The ne di e si y e ec inc eased wi h
ime (p- alue = 0.001) and i was mainly d i en by a posi-
i e complemen a i y e ec , wi h a much lowe and nega i e
selec ion e ec (Table S3). Di e si y e ec s changed wi h sce-
na io (ne di e si y e ec , p- alue = 0.014; complemen a i y
e ec , p- alue = 0.007; selec ion e ec , p- alue = 0.010),
wi h ne di e si y and complemen a i y e ec s being highe
in scena io 2 han in scena io 3 (Table S3, Figu e S1).
Fungal Biomass
Fungal biomass was no signi ican ly a ec ed by lea li e
ype (p- alue = 0.422), al hough i ended o be highe in
heal hy alde and popla a he la e s ages o he expe imen
(Table S3, Fig 2A); and i did no a y among scena ios
(p- alue = 0.384). Howe e , ungal biomass dec eased
wi h ime in all scena ios (p- alue = 0.050), wi h a sha pe
dec ease in scena ios 2 and 3 han in scena io 1 (Table S3,
Fig.2B), al hough i inc eased o did no change wi h ime
in monocul u es. The ne di e si y e ec on ungal biomass
was mainly d i en by a complemen a i y e ec , wi h no di -
e ences among scena ios ei he o he ne di e si y e ec
(p- alue = 0.280) o he complemen a y e ec (p- alue
= 0.436), bu a weak di e ence o he selec ion e ec
(p- alue = 0.014) (Table S3, Fig. S2). The ne di e si y
e ec changed wi h ime, om null o sligh ly posi i e a
he i s sampling da e (10–16% highe han expec ed; Fig.
S2A), o nega i e a he end o he expe imen (Fig. S2D)
(p- alue < 0.001; Table S3). Despi e he non-signi ican di -
e ences among scena ios a he end o he expe imen , i
was 41% lowe han expec ed in scena io 3 and 9% lowe
han expec ed in scena io 1 (Fig. S2D). The ne di e si y
e ec was d i en by a complemen a i y e ec , which p e-
sen ed he same pa e n, shi ing wi h ime om sligh ly
Fig. 1 Decomposi ion a e
(mean ± s.e.; n=9) o lea
li e o i e monocul u es (A)
and h ee mix u es (scena ios;
B). Di e en le e s indica e
signi ican di e ences among
lea li e ypes o scena ios,
analyzed wi h Tukey’s es s

A.Alonso e al. 32 Page 6 o 11
posi i e (Fig. S2B) o nega i e alues (Fig. S2E) (p- alue
< 0.001; Table S3). The selec ion e ec was also a ec ed
by ime (p- alue = 0.032), emaining null in scena io 1 and
nega i e in scena ios 2 and 3 (Table S3, Fig. S2C, Fig. S2F).
Fungal Conidial P oduc ion andAssemblage
S uc u e
Spo ula ion a es a ied wi h lea li e ype (p- alue
< 0.001), ime (p- alue < 0.001), and hei in e ac ion
(p- alue < 0.001) (Table S3); hey we e signi ican ly highe
in in ec ed alde han in ash, popla , and black locus , and
lowes in ash (Fig.3A). The scena ios also di e ed in spo u-
la ion a es (p- alue = 0.001; Table S3), being highe in
scena ios 1 and 2 han in scena io 3 (Fig.3B). Species ich-
ness was a ec ed by lea li e ype (p- alue = 0.005), ime
(p- alue = 0.038), and hei in e ac ion (p- alue = 0.008)
(Table S3), being lowe a day 42 han a day 28. Howe e ,
di e ences among lea li e ypes we e obscu ed by he
in e ac ion wi h ime. Species ichness was also a ec ed by
he scena io (p- alue = 0.017; Table S3), bu pos hoc es s
did no iden i y di e ences, only a end o highe ichness
in scena io 2 (11 species) compa ed o scena ios 1 and 3 (9
species in each) (Table2).
Fungal assemblage s uc u e a ied depending on he
lea li e ype, ime, and hei in e ac ion (p- alues < 0.001;
Table S4). Each lea li e ype showed a di e en esponse
o ime and assemblages in he hi d sampling da e signi i-
can ly di e ed om hose in he i s and second da es; e.g.,
T iscelopho us acumina us Nawawi inc eased (p- alue <
0.010). Despi e a ia ions due o he in e ac ion wi h ime,
pos hoc es s showed signi ican di e ences among all lea
li e ypes, excep be ween heal hy and in ec ed alde and
be ween heal hy alde and popla . Assemblages in popla
and heal hy and in ec ed alde lea li e we e domina ed
by A iculospo a e acladia Ingold (p- alue < 0.001), ol-
lowed by Ala ospo a acumina a Ingold (p- alue = 0.003),
and di e ed by he high ela i e con ibu ion o Te achae-
um elegans Ingold (p- alue = 0.007) in popla , he high
ela i e con ibu ion o Lunulospo a cu ula Ingold (p- alue
= 0.021) in heal hy alde , and a high ela i e con ibu ion o
T. acumina us (p- alue = 0.005) in in ec ed alde . In ec ed
alde was also cha ac e ized by he occu ence o Flagel-
lospo a cu ula Ingold (p- alue = 0.006) and Ala ospo a
pulchella Ma ano á (p- alue = 0.013), wo o i e imes
mo e abundan han in he o he lea li e ypes (Table2).
In black locus , T. acumina us (p- alue = 0.005) was also
dominan . Ash had lowe o al spo ula ion, included T. ele-
gans as he dominan species (p- alue = 0.007), and showed
Fig. 2 Fungal biomass (mean ±
s.e.; n=3) associa ed wi h lea
li e o monocul u es (A) and
mix u es (scena ios; B) a days
14 and 42
Fig. 3 Fungal spo ula ion a es (mean ± s.e.; n=3) associa ed wi h lea li e o monocul u es (A) and mix u es (scena ios; B) a days 14, 28, and 42
Dieback andReplacemen o Ripa ian T ees May Impac S eam Ecosys em Func ioning Page 7 o 11 32
high abundance o Anguillospo a ili o mis G ea h (p- alue
< 0.001) and A. e acladia (Fig.4A, Table2).
Assemblage s uc u e also a ied wi h scena io,
ime, and hei in e ac ion (p- alues ≤ 0.002; Table S4).
Assemblages in he las sampling da e di e ed signi i-
can ly om hose in he i s and second da es and we e
cha ac e ized by a highe p opo ion o T. acumina us
(p- alue < 0.001). Assemblages in scena ios 1 and 2 di -
e ed om hose in scena io 3 (Table S4), because o al
spo ula ion was highe and A. e acladia, he dominan
species, also p esen ed a highe spo ula ion in scena ios
1 and 2 (p- alue = 0.046), wi h A. ili o mis showing
a highe ela i e con ibu ion in scena io 3 (p- alue =
0.046) (Fig.4B, Table2).
Table 2 Speci ic ungal spo ula ion a es (mean ac oss sampling
da es; n=3) in lea li e o i e monocul u es and h ee mix u es
(scena ios). Accumula ed species ichness is gi en a he bo om o
he able. Scena io 1: F axinus angus i olia + Populus nig a + Alnus
lusi anica heal hy; scena io 2: F axinus angus i olia + Populus nig a
+ Alnus lusi anica in ec ed; scena io 3: F axinus angus i olia + Pop-
ulus nig a + Robinia pseudoacacia
Fungal species F axinus
angus i-
olia
Populus nig a Alnus
lusi anica
heal hy
Alnus
lusi anica
in ec ed
Robinia
pseudoa-
cacia
Scena io 1 Scena io 2 Scena io 3
Ala ospo a acumina a Ingold 3.35 24.67 26.76 44.81 6.66 10.08 13.77 5.98
Ala ospo a pulchella Ma ano á 0.25 1.27 0.28 0.28
Anguillospo a ili o mis G ea h. 10.96 3.89 2.90 3.61 0.69 4.54 9.14 4.11
A iculospo a e acladia Ingold 25.77 149.64 141.99 121.37 47.12 147.36 125.31 67.70
Flagellospo a cu ula Ingold 0.62 0.84 1.39 5.28 2.08 2.52 3.49 0.55
Hyd ocina chae ocladia Scheue 0.12 0.91 0.30 0.59 0.21 2.40 3.20 0.32
Lunuslospo a cu ula Ingold 0.20 6.11 5.20 2.71 0.40 13.78 28.27 4.94
Neonec ia lugdunensis (Sacc. &
The y) L. Lomba d & C ous
0.03 0.31
S enocladiella neglec a (Ma ano á
& Descals) Ma ano á & Descals
0.11 0.22 0.73 1.26 0.95 0.10
Taeniospo a g acilis Ma ano á 0.10 0.48 0.07
Te achae um elegans Ingold 49.92 31.85 16.80 7.05 6.11 23.16 47.57 24.85
T iscelopho us acumina us Nawawi 11.13 9.60 22.93 92.60 84.36 45.49 66.34 20.29
Accumula ed species ichness 10 9 10 11 10 9 11 9
Fig. 4 Fungal spo ula ion a es (a e age ac oss sampling da es; n=3) associa ed wi h lea li e o monocul u es (A) and mix u es (scena ios;
B). “O he s” include species con ibu ing less han 5% o o al conidial p oduc ion
A.Alonso e al. 32 Page 8 o 11
Discussion
Ou esul s e ealed changes in s eam ecosys em unc-
ioning and associa ed ungal assemblages ollowing
in ec ion o ipa ian alde by P. ×alni and subsequen
eplacemen o diseased ees by he in asi e black locus .
Ra es o lea li e decomposi ion, a undamen al p ocess
o en used o assess ecological impac s o s esso s on
ecosys em unc ioning [44, 45], expe ienced i s a weak
inc ease a e alde in ec ion and subsequen ly a s ong
dec ease a e eplacemen o in ec ed alde wi h black
locus , when measu ed inlea li e mix u es also con-
aining o he na i e species (i.e., ash and popla ). These
changes had he expec ed di ec ion, con i med hypo heses
i and ii, and e lec ed di e ences in lea li e chemis y.
Lea li e o in ec ed alde showed highe nu ien concen-
a ions and labili y han ha o heal hy alde , as epo ed
be o e [24], which has been a ibu ed o educed nu ien
eso p ion e iciency o in ec ed ees [46] o o accumu-
la ion o nu ien s in lea es due o s ess induced by oo
damage [47]. In con as , black locus lea li e had lowe
N concen a ion han ha o alde and he lowes P concen-
a ion, as well as high polyphenol concen a ions, which
educed i s pala abili y and hence i s decomposi ion a e,
as shown also in o he s udies [33–35, 48].
Despi e he clea di e ences in lea li e chemis y and
he ac ha ends in lea li e decomposi ion ollowed he
expec ed pa e n, he educ ion in decomposi ion a es when
compa ing mix u es wi h heal hy alde (scena io 1) wi h
mix u es wi h black locus (scena io 3) was no s a is ically
signi ican du ing ou expe imen , which las ed 42 days. In
con as , ungal spo ula ion a es we e signi ican ly lowe
in mix u es wi h black locus han in hose wi h heal hy
alde , which was likely caused by he high concen a ion o
polyphenols in black locus , which a e known o ha e an i-
mic obial ac i i y [12, 34, 49, 50]. The lowe ep oduc ion
a e o mic obial decompose s in mix u es wi h black locus
sugges s ha decomposi ion a es migh be signi ican ly
educed i measu ed u he in he longe e m. This is sup-
po ed by he educ ion in ungal biomass ha occu ed wi h
ime in mix u es wi h black locus , bu no in mix u es wi h
heal hy alde . I should be conside ed ha he disappea ance
o alde gene ally occu s a e a pe iod when heal hy alde is
in ec ed. Thus, while decomposi ion and ungal spo ula ion
a es o in ec ed alde lea li e we e sligh ly highe han
hose o heal hy alde lea li e (in monocul u es and in
scena io 1 s. 2), his inc ease was la ge enough o ob ain a
signi ican educ ion in decomposi ion and spo ula ion a es
when in ec ed ees (scena io 2) we e u he eplaced by
black locus (scena io 3).
All lea li e mix u es showed a posi i e ne di e si y
e ec on decomposi ion, ha is, hey decomposed as e
han expec ed based on he decomposi ion o indi idual
species. The ne di e si y e ec was mainly d i en by a
complemen a i y e ec , as shown o he decomposi ion
o o he lea li e mix u es [35, 51], and i inc eased wi h
ime, also as obse ed p e iously [52]. Alde lea li e has
been epo ed o enhance decomposi ion o mo e ecalci-
an lea li e in mix u es [12, 51, 53], ei he by a ac ing
de i i o es [54] o by inc easing he lea nu ien con-
cen a ion o o he species h ough nu ien ans e by
ungal hyphae [55]. In ou expe imen , he e could ha e
been ans e o N om alde o black locus o he o he
species, and also a ans e o P in he opposi e di ec-
ion. This could possibly occu h ough up ake o leached
nu ien s [56, 57], since lea discs we e indi idually in
cons an mo emen in he mic ocosms, mos likely p e-
cluding nu ien ans e by ungal hyphae.
In con as o he pa e n e ealed o decomposi ion,
we ound no signi ican di e ences among lea li e mix-
u es o ungal biomass, wi h only a weak end owa ds
highe biomass in heal hy alde . This con as s wi h p e-
ious s udies, whe e in ec ed alde showed highe un-
gal biomass han heal hy alde [24]. He e, in ec ed alde
showed simila alues o hose o black locus , which usu-
ally p esen s low ungal biomass due o hei low nu ien
and high polyphenol concen a ions [34]. Ou obse ed
pa e n could be caused by nu ien ans e among lea li -
e ypes, which is suppo ed by he posi i e complemen-
a i y e ec on ungal biomass ound a he ea ly s ages o
he expe imen . Towa ds la e s ages, howe e , he e was
a nega i e complemen a i y e ec , sugges ing ha inhibi-
o y compounds p esen in di e en lea li e ypes could
ha e educed ungal g ow h [53, 54], maybe in pa allel o
a deple ion o nu ien s [58]. The g ea e educ ion in mix-
u es wi h black locus and in ec ed alde may be caused
by he highe polyphenol and lowe nu ien concen a ion
in black locus , which make hem mo e p one o nega-
i e e ec s [34], and he enhancemen o decomposi ion
caused by in ec ed alde , which may ha e caused a as e
consump ion o nu ien s [58], which he e o e may no
ha e compensa ed he nega i e e ec s o annins and o he
compounds [12].
Lea li e o di e en species showed di e en ungal
assemblages, acco ding o he di e en subs a e p e e -
ences shown by ungal species [11]. As p e iously obse ed
[24], heal hy and in ec ed alde showed simila assem-
blages, bu mix u es wi h alde had di e en assemblages
compa ed o mix u es wi h black locus . This di e ence was
mainly d i en by A. e acladia, which was he mos abun-
dan hyphomyce e in all mix u es, bu i was educed in he
p esence o black locus . This p obably occu ed because
i s spo ula ion was limi ed due o lowe nu ien and highe
polyphenol concen a ions [14, 54, 59, 60].
Dieback andReplacemen o Ripa ian T ees May Impac S eam Ecosys em Func ioning Page 9 o 11 32
Conclusions
Ou s udy e eals changes in he key s eam ecosys em p o-
cess o lea li e decomposi ion and associa ed ungal assem-
blages, ollowing a sequence o al e a ions in species compo-
si ion o he ipa ian o es . The mos s iking changes occu
when alde is in ec ed by P. ×alni, which sligh ly accele a es
he p ocess o lea li e decomposi ion i compa ed o non-
in ec ed alde ees andwhich, a e he eplacemen o alde
wi h he exo ic black locus , induces a signi ican educ ion
in he decomposi ion p ocess and a subs an ial change in he
cha ac e is ics o mic obial decompose assemblages. Ou
esul s also sugges ha changes de ec ed 42 days a e he
expe imen s a ed would be in ensi ied in he longe e m, as
p oposed elsewhe e [24]. Impac s o he ipa ian o es , such
as in ec ious diseases—which d i e changes in he ai s o
na i e species and can ul ima ely lead o hei loss—and spe-
cies in asions—which in oduce species wi h di e en ai s
om hose o na i e species—ha e he capaci y o al e he
unc ioning and s uc u e o he s eam ecosys em. Ou s udy
shows how his occu s e en i he na i e and in asi e species
belong o he same unc ional g oup, in his case N- ixing
species. O e all, ou s udy highligh s he impo ance o p e-
se ing na i e and heal hy ipa ian ege a ion in o de o
main ain p ope ecosys em unc ioning.
Supplemen a y In o ma ion The online e sion con ains supplemen-
a y ma e ial a ailable a h ps:// doi. o g/ 10. 1007/ s00248- 024- 02343-w.
Acknowledgemen s E gos e ol analyses we e o de ed o Ins i u o do
Ambien e Tecnologia e Vida (IATV, Uni e si y o Coimb a, Po u-
gal).Commen s p o ided by wo anonymous e iewe s on an ea lie
e sion o he manusc ip we e much app ecia ed.
Au ho Con ibu ion All au ho s concei ed he ideas and designed
me hodology; AA and VF collec ed he da a and analyzed he da a;
AA led he w i ing o he manusc ip . All au ho s con ibu ed c i ically
o he d a s and ga e inal app o al o publica ion.
Funding Open Access unding p o ided hanks o he CRUE-CSIC
ag eemen wi h Sp inge Na u e. This s udy was inanced by he Po u-
guese Founda ion o Science and Technology (FCT) h ough p ojec s
UIDP/04292/2020 and UIDB/04292/2020 g an ed o MARE, p ojec
LA/P/0069/2020 g an ed o he Associa e Labo a o y ARNET, and
inancial suppo g an ed o VF (CEECIND/02484/2018); by Basque
Go e nmen unds o Consolida ed Resea ch G oups g an ed o LB
(Re . IT1471-22); and by a UPV/EHU p edoc o al ellowship g an ed
o AA. AS was g an ed by ACCIÓN VI-23 o a esea ch s ay unde -
aken in July 2023 a MAREFOZ, Figuei a da Foz, Po ugal. Alde lea
li e sampling and pos ing we e unded by g an AGL2014-53822-C2-
1-R om he Spanish Minis y o Economy and Compe i i eness and
he Eu opean Union’s Eu opean Regional De elopmen Fund (ERDF).
Da a A ailabili y Da a is a ailable om he Open Science F amewo k
Reposi o y.
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