Ci a ion: Mo illas, L.; Roales, J.; C uz,
C.; Munzi, S. Non-Toxic Inc eases in
Ni ogen A ailabili y Can Imp o e
he Abili y o he Soil Lichen Cladonia
angi e ina o Cope wi h
En i onmen al Changes. J. Fungi
2022,8, 333. h ps://doi.o g/
10.3390/jo 8040333
Academic Edi o : Saman ha
C. Ka una a hna
Recei ed: 8 Feb ua y 2022
Accep ed: 16 Ma ch 2022
Published: 23 Ma ch 2022
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Fungi
Jou nal o
A icle
Non-Toxic Inc eases in Ni ogen A ailabili y Can Imp o e he
Abili y o he Soil Lichen Cladonia angi e ina o Cope wi h
En i onmen al Changes
Lou des Mo illas 1,* , Ja ie Roales 1,2 , C is ina C uz 1and Sil ana Munzi 1,3
1Cen e o Ecology, E olu ion and En i onmen al Changes, Faculdade de Ciências, Uni e sidade de Lisboa,
Campo G ande, Bloco C2, 1749-016 Lisbon, Po ugal; ja ie [email p o ec ed] (J.R.); [email p o ec ed] (C.C.);
[email p o ec ed] (S.M.)
2
Depa amen o de Sis emas Físicos, Químicos y Na u ales, Uni e sidad Pablo de Ola ide, C a. U e a Km 1,
41013 Se ille, Spain
3Cen o In e uni e si á io de His ó ia das Ciências e da Tecnologia Faculdade de Ciências, Uni e sidade de
Lisboa, Campo G ande, 1749-016 Lisbon, Po ugal
*Co espondence: [email p o ec ed]
Abs ac :
Clima e change and a mosphe ic ni ogen (N) deposi ion on d ylands a e g ea ly h ea -
ening hese especially ulne able a eas. Soil bioc us - o ming lichens in d ylands can p o ide ea ly
indica o s o hese dis u bances and play a pi o al ole, as hey con ibu e o key ecosys em se ices.
In his s udy, we explo ed he e ec s o di e en long- e m wa e a ailabili y egimes simula ing
clima e changes and hei in e ac ion wi h N addi ion on he physiological esponse o he soil
lichen Cladonia angi e ina. Th ee se s o his lichen we e subjec ed o con ol, educed wa e ing, and
educed wa e ing and N addi ion (40 kg NH
4
NO
3
ha
−1
yea
−1
) ea men s o 16 mon hs. Finally,
all samples we e subjec ed o daily hyd a ion cycles wi h N-en iched wa e a wo le els (40 and 80
kg
NH4NO3ha−1yea −1
) o 23 days. We ound ha educed wa e ing signi ican ly dec eased he
i ali y o his lichen, whe eas N addi ion unexpec edly helped lichens subjec ed o educed wa e ing
o cope wi h s ess p oduced by high empe a u es. We also ound ha long- e m exposu e o N
addi ion con ibu ed o he acclima ion o highe N a ailabili y. O e all, ou da a sugges ha he
in e ac ions be ween educed wa e ing and inc eased N supply and empe a u e ha e an impo an
po en ial o educe he physiological pe o mance o his soil lichen.
Keywo ds:
soil lichens; soil bioc us ; global change; clima e change; biomoni o ing; syne ge ic e ec s;
educed wa e ing; a idi y; d ylands; Medi e anean ecosys ems
1. In oduc ion
D ylands (a id, semia id, and d y subhumid egions ecei ing in equen p ecipi-
a ions) co e almos 45% o he e es ial su ace, comp ising Ea h’s la ges e es ial
biome [
1
]. These a eas a e highly ulne able o dese i ica ion and global change [
2
,
3
], and
a e expec ed o expand due o he p ojec ed clima e wa ming [
4
]. Scien is s o esee ha
hese ecosys ems will espond o he u u e clima e wi h impo an shi s in communi y
composi ion and biogeochemical cycles [
5
,
6
], wi h global implica ions o ca bon (C) and
nu ien cycling [
7
,
8
]. Despi e hese ac s, li e a u e ela ed o global change is domina ed
by s udies pe o med in o he ecosys ems [
1
], lea ing impo an ques ions on how key
ecosys em p ocesses in d ylands will espond o global change un esol ed.
Ni ogen (N) and wa e a e he main ac o s limi ing p ima y p oduc ion in d yland
ecosys ems [
9
], ende ing al e a ions o hei a ailabili y highly likely o cause communi y-
le el impac s. The cu en con ex o clima e change is p ojec ed o cause ele an changes
in ain all dynamics in d ylands, inc easing dese i ica ion and d ough s [
10
]. This scena io
u ges us o unde s and how such al e a ions in highly ulne able a eas will ansla e in o
J. Fungi 2022,8, 333. h ps://doi.o g/10.3390/jo 8040333 h ps://www.mdpi.com/jou nal/jo
J. Fungi 2022,8, 333 2 o 13
dis u bed ecosys em unc ioning. An e en mo e p essing issue is o e eal how in e ac ions
be ween hese ac o s and o he d i e s o global change such as a mosphe ic pollu an s
migh a ec esponses. In e ac ions can igge complex and unce ain esponses [
11
,
12
],
a ec ing he ecosys em dynamics [
13
]. The N deposi ion in bo h oxidized (NO
x
) and
educed (NH
3
) o ms is inc easing globally due o human-induced ac i i ies [
14
]. In
d yland ecosys ems loca ed in he Medi e anean Basin, N inpu s a e expec ed o inc ease
om he 7 kg N ha
−1
yea
−1
o he mid-1990s o 12 kg N ha
−1
yea
−1
in 2050 [
15
]. The abili y
o disen angle he linkages be ween N deposi ion a es, clima e change and ecosys em
e ec s o pi o al d yland communi ies would subs an ially ad ance ou capaci y o assess
and p edic bio ic esponses o global change.
D ylands h oughou he wo ld ea u e spa se ege a ion co e su ounded by a
ma ix de oid o ascula plan s [
16
]. This ma ix is equen ly colonized by bioc us com-
muni ies domina ed by cyanobac e ia, mosses and lichens, ha ep esen a pi o al bio ic
componen o hese a eas p o iding nume ous key ecosys em se ices [
17
,
18
]. Lichens,
which cons i u e one o he main componen s o hese soils’ bioc us s, a e ulne able
o a mosphe ic changes, which makes hem highly e icien ea ly indica o s o bo h N
deposi ion [19] and changes in clima e [20].
As poikilohyd ic o ganisms, lichens ole a e apidly al e na e pe iods o deep desicca-
ion wi h di e en le els o hyd a ion, being able o eac i a e hei me abolic ac i i y in
esponse o small amoun s o wa e [
21
]. The absence o ain e en s du ing he summe
mon hs is a ypical ea u e o d ylands. Howe e , ha does no imply ha lichens a e
inac i e du ing hose long pe iods. Lichens which ha e ebouxioid algae as pho obion s,
as is he case o Cladonia angi e ina, can eco e wi h no only ain o dew, bu also wi h a
high deg ee o ai humidi y [
22
,
23
]. Lichens ea u e a ci cadian cycle o me abolic ac i i y
linked o ai humidi y le els [
24
,
25
]. This mechanism allows hem o become ac i e a e
sunse , esponding o dec eased empe a u es, and emain ac i e up o sun ise, when
hey dehyd a e p og essi ely because o he inc eased empe a u e and dec eased ai (and
hallus) humidi y. This ci cadian hyd a ion–dehyd a ion cycle may be a ec ed by he
inc eased a idi y p ojec ed o hese a eas in such a way ha inc eased empe a u e and
dec eased ai humidi y will p e en lichens om being ac i a ed by nigh on daily basis.
Thus, expec ed changes in ain all and empe a u e can s ongly impac he unc ioning
o soil lichens o ming he bioc us [
26
] and he c i ical ecosys em p ocesses a ec ed by
hem [27,28].
Ni ogen is an essen ial nu ien o lichens, playing a c ucial ole o many p ocesses
o bo h he pho obion and he mycobion . Howe e , N can be a s ess ac o i supplied
in excess [
29
,
30
] and i has been ela ed o shi s o lichen communi ies [
31
]. Gi en he
signi icance o hese o ganisms and he p ocesses hey help con ol, he e is a g owing body
o li e a u e on how lichens will espond o he expec ed exace ba ed N deposi ion [
32
–
34
].
Howe e , i is la gely unknown how he e ec s o N deposi ion will in e ac wi h hose
o clima e change when impac ing soil lichens. Thus, he abili y o assess he syne gis ic
e ec s o hese en i onmen al changes h ough lichen- o ming bioc us s would signi ican ly
inc ease ou unde s anding o d yland esilience o global change.
Ou goals we e o (i) explo e he e ec s o di e en long- e m wa e a ailabili y
egimes simula ing clima e changes and hei in e ac ion wi h N addi ion (40 kg h
−1
yea
−1
NH
4
NO
3
) on he physiological esponse o he soil bioc us - o ming lichen Cladonia angi e-
ina and (ii) assess i a p io long pe iod o exposu e o di e en s esses de e mines he
abili y o hese lichens o deal wi h inc eased N addi ion (40 and 80 kg h
−1
yea
−1
NH
4
NO
3
).
We hypo hesized ha he syne gis ic e ec o inc eased a idi y and N addi ion would a ec
he physiological pe o mance o Cladonia. The e o e, we expec ed (i) a lowe i ali y in
N- ea ed samples exposed o educed wa e ing han in hose exposed o one s esso only.
We also expec ed ha (ii) he long- e m exposu e o N addi ion would ha e con ibu ed
o he acclima ion o highe N a ailabili y h ough ac i a ion o esponse mechanisms
o e he pe iod. To add ess hese hypo heses, we pe o med a manipula i e mic ocosm
expe imen o e 16 mon hs in which he e ec o educed wa e ing and he in e ac ion
J. Fungi 2022,8, 333 3 o 13
be ween educed wa e ing and N deposi ion on he soil lichen Cladonia angi e ina we e
es ed. We used chlo ophyll a luo escence as a sensi i e bu non-des uc i e app oach o
show how pho osyn he ic o ganisms espond o en i onmen al s esso s [
35
]. Disen an-
gling he e ec s o mul iple global change d i e s on hese sensi i e elemen s o ecosys ems
is pa amoun o imp o ing he use o lichens as bioindica o s o p o ec ing ulne able
biomes and p ojec ing he po en ial consequences on ecosys em unc ioning.
2. Ma e ials and Me hods
2.1. Lichen Sampling
In No embe 2019, 50 samples o Cladonia angi e ina we e collec ed om a Medi e -
anean maquis (see de ailed desc ip ion in Dias e al. [
36
]) wi h a well-de eloped bioc us
communi y loca ed in Se a da A ábida, in he A ábida Na u al Pa k, sou h o Lisbon,
Po ugal (38
◦
29
0
24.2
00
N, 9
◦
01
0
58.0
00
W, Figu e S1). Backg ound N deposi ion a he si e
is
5.2 kg ha−1yea −1
(2.9 kg NO
x
+ 2.3 kg NH
y
) [
37
]. Lichens we e de ached om he
subs a e and anspo ed o he labo a o y whe e hey we e ca e ully cleaned o emo e
any impu i ies. All lichens we e s o ed in ays in a well-li a ea a oom empe a u e in
and we e ully ehyd a ed be o e pe o ming any measu emen s.
2.2. Incuba ion
The incuba ion was pe o med indoo s in a well ae a ed loca ion and absen om hea
o cold sou ces, wi h empe a u e oscilla ing mode a ely acco ding o ou doo wea he .
Lichen samples we e con ained in 50 cus om-buil wi e mesh cages (Figu e S2), each wi h
app oxima ely 4.5 g o lichens. Samples we e ins alled in a e ical s uc u e hanging
om a no h- acing window, ensu ing ha no di ec sunligh eached hem. Fans placed
abo e he samples acili a ed d ying a e ea men . To p e en e ical con amina ion,
ea men s wi h highe N concen a ions and/o wa e ing equencies we e placed a he
bo om o he s uc u e. The posi ion o he samples was changed e e y ew days o ensu e
homogeneous condi ions.
2.3. T ea men s
As lichens ha e a slow g ow h a e, ou expe imen al design accoun ed o a long- e m
ea men o maximize he likelihood o lichen esponse. The expe imen consis ed o wo
consecu i e phases (Figu e 1). Du ing he i s phase, which las ed 16 mon hs, we applied
h ee di e en ea men s, each o a subg oup o samples: con ol, educed wa e ing (RW),
and educed wa e ing and N deposi ion (RW+N). Con ol samples we e subjec ed o one
daily a i icial wa e ing cycle, samples es ing he e ec o educed wa e ing we e wa e ed
wice pe week and samples accoun ing o he in e ac ion be ween educed wa e ing and
N deposi ion we e wa e ed wi h N-en iched wa e wice pe week (Figu e 1). T ea men s
in his phase in ol ed he imme sion o he samples in mine al wa e , in he case o con ol
and RW samples, o in N-en iched wa e (equi alen o a deposi ion o 40 kg N h
−1
yea
−1
,
N40), in he case o RW+N samples, o 30 s. Following his long- e m ea men , in he
second phase o ou expe imen , all samples we e subjec ed o daily hyd a ion cycles wi h
N-en iched wa e a wo le els: 40 and 80 kg N h
−1
yea
−1
(N40 and N80, he ea e ) o
23 days
(Figu e 1). As luo escence was moni o ed on a daily basis du ing his phase, i
las ed long enough o lichens o espond o ou sho - e m ea men , and he expe imen
ended when we ound di e ences be ween ea men s. T ea men s consis ed o a 30
s imme sion o he samples in ei he a 17.15 mg/L o a 34.3 mg/L NH
4
NO
3
solu ion,
co esponding o N40 and N80. Mine al wa e wi h low mine al con en was used o a oid
osmo ic shock on lichen issues. The chosen N doses in his expe imen we e lowe han
he N deposi ion epo ed o o he a eas in Medi e anean- ype ecosys ems (145 kg N
ha
−1
yea
−1
[
38
,
39
]), bu high enough o simula e he u u e p ojec ions o N deposi ion in
his ype o habi a . The concen a ion o N-en iched wa e was compu ed using a e age
yea ly p ecipi a ion o calcula e inal solu ion concen a ions. All wa e ing ea men s we e
applied a a ound 11 AM CET so ha pho osyn he ic p ocesses we e ac i e du ing dayligh .
J. Fungi 2022,8, 333 4 o 13
The numbe o eplica es a ied among ea men s and phases, bu in all cases a minimum
o 5 eplica es was used.
J. Fungi 2022, 8, x FOR PEER REVIEW 4 o 13
yea ly p ecipi a ion o calcula e inal solu ion concen a ions. All wa e ing ea men s
we e applied a a ound 11 AM CET so ha pho osyn he ic p ocesses we e ac i e du ing
dayligh . The numbe o eplica es a ied among ea men s and phases, bu in all cases a
minimum o 5 eplica es was used.
Figu e 1. G aphical ep esen a ion o he expe imen al design (see ex o de ails). Do s indica e
he equency o daily a i icial wa e ings. Con ol: samples subjec ed o one daily a i icial wa e -
ing cycle (blue do s). RW: samples wa e ed wice pe week ( ed do s). RW+N: samples wa e ed
wi h N-en iched wa e wice pe week (black do s). Du ing Phase 2, -N40 (black do s) and -N80
(o ange do s) indica e he daily hyd a ion o he samples wi h N-en iched wa e a wo le els: 40
and 80 kg N h−1y−1.
2.4. Chlo ophyll a Fluo escence
A Handy PEA plan e iciency analyze (Hansa ech Ins umen s LTD, Pen ney, UK)
was used o quan i y ea men e ec s h ough he de e mina ion o he F /Fm a io, he
mos equen ly used chlo ophyll a luo escence pa ame e in ecological esea ch (e.g.,
Raggio e al. [40]; Mo illas e al. [32]). The F /Fm a io is used as an indica o o he pho-
obion i ali y and a p o ide o an ea ly wa ning o physiological s ess [41]. Ten
minu es a e each wa e ing ea men , once he samples we e ully ehyd a ed, lichens
we e da k-adap ed a oom empe a u e o 15 min o maximize oxida ion o he p ima y
quinone elec on accep o o PSII. Then, chlo ophyll a luo escence was measu ed.
2.5. S a is ical Analyses
Da a we e checked o con o mi y wi h epea ed measu es analysis o a iance
(ANOVA) assump ions using Shapi o–Wilk no mali y es and Mauchly’s es o sphe ic-
i y. No mali y es s o he esiduals by ime poin indica ed ha hey ollowed app oxi-
ma ely a no mal dis ibu ion. When sphe ici y could no be ensu ed, a G eenhouse–
Geisse co ec ion was used. Di e ences in F /Fm alues among samples subjec ed o di -
e en ea men s we e e alua ed ia epea ed measu es p ocedu e ollowing a 2-way
mixed ANOVA design wi h 1 wi hin-subjec s ac o and 1 be ween-g oups ac o in IBM
SPSS S a is ics 23.0 (SPSS Inc., Chicago, IL, USA). Pai wise compa isons we e pe o med
Figu e 1.
G aphical ep esen a ion o he expe imen al design (see ex o de ails). Do s indica e he
equency o daily a i icial wa e ings. Con ol: samples subjec ed o one daily a i icial wa e ing
cycle (blue do s). RW: samples wa e ed wice pe week ( ed do s). RW+N: samples wa e ed wi h
N-en iched wa e wice pe week (black do s). Du ing Phase 2, -N40 (black do s) and -N80 (o ange
do s) indica e he daily hyd a ion o he samples wi h N-en iched wa e a wo le els: 40 and
80 kg N h−1yea −1.
2.4. Chlo ophyll a Fluo escence
A Handy PEA plan e iciency analyze (Hansa ech Ins umen s LTD, Pen ney, UK)
was used o quan i y ea men e ec s h ough he de e mina ion o he F /Fm a io, he
mos equen ly used chlo ophyll a luo escence pa ame e in ecological esea ch (e.g.,
Raggio e al. [
40
]; Mo illas e al. [
32
]). The F /Fm a io is used as an indica o o he
pho obion i ali y and a p o ide o an ea ly wa ning o physiological s ess [
41
]. Ten
minu es a e each wa e ing ea men , once he samples we e ully ehyd a ed, lichens
we e da k-adap ed a oom empe a u e o 15 min o maximize oxida ion o he p ima y
quinone elec on accep o o PSII. Then, chlo ophyll a luo escence was measu ed.
2.5. S a is ical Analyses
Da a we e checked o con o mi y wi h epea ed measu es analysis o a iance
(ANOVA) assump ions using Shapi o–Wilk no mali y es and Mauchly’s es o sphe ici y.
No mali y es s o he esiduals by ime poin indica ed ha hey ollowed app oxima ely
a no mal dis ibu ion. When sphe ici y could no be ensu ed, a G eenhouse–Geisse co -
ec ion was used. Di e ences in F /Fm alues among samples subjec ed o di e en
ea men s we e e alua ed ia epea ed measu es p ocedu e ollowing a 2-way mixed
ANOVA design wi h 1 wi hin-subjec s ac o and 1 be ween-g oups ac o in IBM SPSS
S a is ics 23.0 (SPSS Inc., Chicago, IL, USA). Pai wise compa isons we e pe o med by com-
pa ing main e ec s h ough pos hoc es s using he Bon e oni co ec ion. To in es iga e
in e ac ions, da a we e di ided in o subse s acco ding o he ea men s and hen we e
subjec ed o epea ed-measu es analyses.
J. Fungi 2022,8, 333 5 o 13
3. Resul s
3.1. E ec s o Di e en Long-Te m Wa e Regimes and Thei In e ac ion wi h N Pollu ion
Compa ison be ween Con ol and RW samples assessed he e ec s o di e en long-
e m wa e egimes on Cladonia i ali y (Figu e 2). Signi ican e ec s o he educed wa e ing
ea men s and ime we e de ec ed (Figu e 2and Table 1). Indeed, we ound a gene al
end o dec eased physiological pe o mance in all samples subjec ed o educed wa e ing
(bo h exposed and no exposed o N addi ion), al hough signi ican di e ences we e
only ound be ween Con ol and RW (p
Con ol/RW
< 0.0001, p
Con ol/RW+N
< 0.141, Figu e 2
and Table 1). The e was a signi ican ea men x ime in e ac ion, which on u he
examina ion showed a signi ican e ec o ime o e e y ea men (Table 1). Fo all
ea men s, bu mainly o hose subjec ed o educed wa e ing, F /Fm alues ollowed a
seasonal end: minimum alues we e eached coinciding wi h a hea wa e in May 2020
and wi h summe ime in July and Augus 2020 (Figu e 2). To es he in e ac i e e ec o
educed wa e ing and N pollu ion, we compa ed RW and RW+N (Figu e 2). Ou esul s
showed ha he physiological pe o mance o lichens subjec ed o hese ea men s was
e y simila and g ea es di e ences we e obse ed when esponding o en i onmen al
s ess p oduced by inc eased empe a u es (Figu e 2). Du ing hese clima ic condi ions, N
addi ion imp o ed lichen i ali y, al hough no signi ican di e ences be ween ea men s
we e ound (p
RW/RW+N
= 0.076, Figu e 2and Table 1). Lowe alues o measu emen s a he
end o he expe imen in he Con ol ea men indica ed de e io a ion in he index due o
ime, i.e., he expe imen al condi ions we e likely a ec ing hese samples disp opo ionally
compa ed o he o he ea men s (RW and RW+N). Bleaching was obse ed in lichens
esponding o he Con ol ea men (Figu e S3).
J. Fungi 2022, 8, x FOR PEER REVIEW 5 o 13
3.1. E ec s o Di e en Long-Te m Wa e Regimes and Thei In e ac ion wi h N Pollu ion
Compa ison be ween Con ol and RW samples assessed he e ec s o di e en long-
e m wa e egimes on Cladonia i ali y (Figu e 2). Signi ican e ec s o he educed wa-
e ing ea men s and ime we e de ec ed (Figu e 2 and Table 1). Indeed, we ound a gen-
e al end o dec eased physiological pe o mance in all samples subjec ed o educed wa-
e ing (bo h exposed and no exposed o N addi ion), al hough signi ican di e ences
we e only ound be ween Con ol and RW (pCon ol/RW < 0.0001, pCon ol/RW+N < 0.141, Figu e 2
and Table 1). The e was a signi ican ea men x ime in e ac ion, which on u he exam-
ina ion showed a signi ican e ec o ime o e e y ea men (Table 1). Fo all ea men s,
bu mainly o hose subjec ed o educed wa e ing, F /Fm alues ollowed a seasonal
end: minimum alues we e eached coinciding wi h a hea wa e in May 2020 and wi h
summe ime in July and Augus 2020 (Figu e 2). To es he in e ac i e e ec o educed
wa e ing and N pollu ion, we compa ed RW and RW+N (Figu e 2). Ou esul s showed
ha he physiological pe o mance o lichens subjec ed o hese ea men s was e y sim-
ila and g ea es di e ences we e obse ed when esponding o en i onmen al s ess p o-
duced by inc eased empe a u es (Figu e 2). Du ing hese clima ic condi ions, N addi ion
imp o ed lichen i ali y, al hough no signi ican di e ences be ween ea men s we e
ound (p RW/RW+N = 0.076, Figu e 2 and Table 1). Lowe alues o measu emen s a he end
o he expe imen in he Con ol ea men indica ed de e io a ion in he index due o
ime, i.e., he expe imen al condi ions we e likely a ec ing hese samples disp opo ion-
ally compa ed o he o he ea men s (RW and RW+N). Bleaching was obse ed in lichens
esponding o he Con ol ea men (Figu e S3).
Figu e 2. Tempo al e olu ion o F /Fm a io du ing phase 1 o he expe imen o Con ol (wa e ed
e e y day, blue squa es), RW (wa e ed wice pe week, ed do s) and RW+N (wa e ed wice pe
week wi h 40 kg N h−1y−1, black iangles) samples. Uppe case le e s indica e signi ican di e ences
among ea men s. The s udy was conduc ed om No embe 2019 o Ma ch 2021.
Figu e 2.
Tempo al e olu ion o F /Fm a io du ing phase 1 o he expe imen o Con ol (wa e ed
e e y day, blue squa es), RW (wa e ed wice pe week, ed do s) and RW+N (wa e ed wice pe week
wi h 40 kg N h
−1
yea
−1
, black iangles) samples. Uppe case le e s indica e signi ican di e ences
among ea men s. The s udy was conduc ed om No embe 2019 o Ma ch 2021.
J. Fungi 2022,8, 333 6 o 13
Table 1.
Repea ed measu es ANOVA analyses o all ea men s du ing phase 1 o ou expe imen .
Con ol = wa e ing e e y day, RW = Reduced wa e ing ( wice pe week) and RW+N = Reduced
wa e ing ( wice pe week) and ni ogen addi ion. d : deg ees o eedom, F: F- alue, p:p- alue.
Fac o d F p
T ea men 2 11.810 <0.0001
Time 7.622 39.155 <0.0001
T ea men ×Time 15.245 5.578 <0.0001
In e ac ion Con ol 4.238 18.835 <0.0001
In e ac ion RW 6.525 27.626 <0.0001
In e ac ion RW+N 3.384 9.713 <0.0001
3.2. E ec s o P io N Exposu e on he Abili y o Cladonia o Deal wi h Inc eased N A ailabili y
Signi ican e ec s o he di e en N ea men s we e ound in phase 2 o ou ex-
pe imen o bo h 40 and 80 kg N h
−1
yea
−1
(Figu e 3, Table 2). Long- e m N addi ion
signi ican ly inc eased F /Fm alues o Cladonia samples when exposed o u he N a ail-
abili y a bo h 40 (Figu e 3a) and 80 (Figu e 3b) kg N h
−1
yea
−1
: RW+N samples showed
he highes i ali y alues. Con ol samples had he lowes alues while RW samples we e
in e media e (Figu e 3a,b). Ni ogen load had no signi ican e ec . F /Fm alues did no
ollow any pa icula pa e n o e ime o any ea men (Figu e 3a,b).
J. Fungi 2022, 8, x FOR PEER REVIEW 6 o 13
Table 1. Repea ed measu es ANOVA analyses o all ea men s du ing phase 1 o ou expe imen .
Con ol = wa e ing e e y day, RW = Reduced wa e ing ( wice pe week) and RW+N = Reduced
wa e ing ( wice pe week) and ni ogen addi ion. d : deg ees o eedom, F: F- alue, p: p- alue.
Fac o d F p
T ea men 2 11.810 <0.0001
Time 7.622 39.155 <0.0001
T ea men × Time 15.245 5.578 <0.0001
In e ac ion Con ol 4.238 18.835 <0.0001
In e ac ion RW 6.525 27.626 <0.0001
In e ac ion RW+N 3.384 9.713 <0.0001
3.2. E ec s o P io N Exposu e on he Abili y o Cladonia o Deal wi h Inc eased N A ailabili y
Signi ican e ec s o he di e en N ea men s we e ound in phase 2 o ou expe i-
men o bo h 40 and 80 kg N h−1y−1 (Figu e 3, Table 2). Long- e m N addi ion signi ican ly
inc eased F /Fm alues o Cladonia samples when exposed o u he N a ailabili y a
bo h 40 (Figu e 3a) and 80 (Figu e 3b) kg N h−1y−1: RW+N samples showed he highes
i ali y alues. Con ol samples had he lowes alues while RW samples we e in e me-
dia e (Figu e 3a,b). Ni ogen load had no signi ican e ec . F /Fm alues did no ollow
any pa icula pa e n o e ime o any ea men (Figu e 3a,b).
Figu e 3. Tempo al changes in F /Fm a io du ing phase 2 o he expe imen ecei ing 40 (a) and 80
(b) kg N h−1y−1. Con ol samples in phase 1 (wa e ed e e y day, blue do s) a e labeled as C-N40 and
C-N80, espec i ely. RW samples in phase 1 (wa e ed wice pe week, ed squa es) a e labeled as
RW-N40 and RW-N80, espec i ely. RW+N samples in phase 1 (wa e ed wice pe week wi h 40 kg
N h−1y−1, black iangles) a e labeled as RW+N40 and RW+N80, espec i ely. Uppe case le e s indi-
ca e signi ican di e ences among ea men s.
Figu e 3.
Tempo al changes in F /Fm a io du ing phase 2 o he expe imen ecei ing 40 (
a
) and 80
(
b
) kg N h
−1
yea
−1
. Con ol samples in phase 1 (wa e ed e e y day, blue do s) a e labeled as C-N40
and C-N80, espec i ely. RW samples in phase 1 (wa e ed wice pe week, ed squa es) a e labeled
as RW-N40 and RW-N80, espec i ely. RW+N samples in phase 1 (wa e ed wice pe week wi h
40 kg N h−1yea −1
, black iangles) a e labeled as RW+N40 and RW+N80, espec i ely. Uppe case
le e s indica e signi ican di e ences among ea men s.
J. Fungi 2022,8, 333 7 o 13
Table 2.
Repea ed measu es ANOVA analyses o all ea men s du ing phase 2 o ou expe imen .
d : deg ees o eedom, F: F- alue, p:p- alue.
Fac o d F p
40 kg N h−1yea −1
(N-40)
T ea men 2 12.293 0.001
Time 11 1.385 0.187
T ea men ×Time 22 1.054 0.406
80 kg N h−1yea −1
(N-80)
T ea men 2 5.566 0.019
Time 11 2.309 0.013
T ea men ×Time 22 0.703 0.830
4. Discussion
In inc easingly a id Medi e anean a eas, wa e is conside ed o be he mos limi ing
en i onmen al ac o o p ima y p oduc ion and mic obial li e [
42
,
43
], despi e he di ec
limi a ions associa ed wi h he low nu ien a ailabili ies ha p e ail in his ecosys em [
44
].
In he case o lichens, educed wa e a ailabili y and high ligh a e conside ed majo s ess
componen s [
45
]. I was he e o e expec ed ha educed wa e a ailabili y would be mos
in luen ial, and ha N addi ion would ha e a seconda y, bu syne gis ic de imen al e ec .
Howe e , while ou s udy showed a nega i e esponse o he soil lichen Cladonia angi e ina
o educed wa e a ailabili y, N addi ion had a posi i e e ec (40 kg N h
−1
yea
−1
), helping
o amelio a e high empe a u e s ess. The e o e, ou da a did no suppo ou i s hypo h-
esis. Howe e , ou indings p o ed ha he long- e m exposu e o N addi ion con ibu ed
o he acclima ion o highe N a ailabili y, suppo ing ou second hypo hesis.
Bioc us lichens in ou expe imen p o ed o be pa icula ly esponsi e o educed
wa e ing. The sensi i i y o lichens o changes in humidi y is ela ed o hei poikilohyd ic
na u e. The wa e con en o hei cells is in equilib ium wi h he su ounding en i onmen
and hei physiological ac i i y is limi ed o pe iods o hyd a ion. Thus, pe o mance and
g ow h du ing each hyd a ion e en a e key ac o s ha will de e mine hei long- e m
su i al. In poikilohyd ic o ganisms, each we ing e en induces a pe iod o ne ca bon
loss due o espi a ion, epa a ion, and eins a emen o me abolism. When pho osyn hesis
s a s, ca bon ixa ion exceeds espi a ion, which leads o ne ca bon gains du ing he we
pe iod. When issues desicca e, pho osyn hesis ceases, leading o a phase o ca bon loss [
46
].
The e o e, in d ylands, each hyd a ion e en in ol es a balance be ween ene gy spen
du ing ehyd a ion and ene gy gained while hyd a ed, which leads o an o e all posi i e
o nega i e ca bon balance [
47
]. When assessing longe ime scales, ca bon balances om
indi idual we ing e en s a e igh ly linked o long- e m g ow h and su i al. The iming
o we ing e en s and he leng h o desicca ion pe iod a e coupled, which de e mines he
lichen ca bon balance. Lichens p og essi ely d ying in ea ly mo ning as a esul o slow
empe a u e ise will likely ha e a mo e posi i e ca bon balance and su i al han lichens
d ying suddenly as a esul o high empe a u e. We belie e ha he declining F /Fm
alues epo ed o he con ol samples a he end o phase 1 in his s udy a e due o he
cumula i e e ec o nega i e ca bon balance esul ing om daily wa e ing. As wa e ing
ea men o con ol samples was applied daily a ound 11:00 CET, ai empe a u e was
likely o exceed hose o which lichens a e exposed ollowing he mo ning dew. This mo e
apid d ying led o a nega i e ca bon balance. In ag eemen , Maphangwa e al. [
48
] ound
ha he highes e ec i e pho osyn he ic quan um yield was measu ed in lichens be o e
10:00 h and he lowes a ound he sola noon. Tempe a u es abo e 20–25
◦
C i e e sibly
dec eased he pho osyn he ic a e in lichens in a id dese s [
49
] and abo e 33
◦
C in a ho
s eam en a ea o Hawaii [
50
]. Six een mon hs o daily wa e ing and po en ially nega i e
ca bon balance a ec ed he i ali y o ou samples, indica ed by he lowe F /Fm alues,
J. Fungi 2022,8, 333 8 o 13
educing hei abili y o ole a e phase 2 o ou expe imen . Damage om apid desicca ion
in o he poikilohyd ic o ganisms has been epo ed o in ol e cell memb ane leakage o
ions and elec oly es and educed chlo oplas and mi ochond ial memb ane in eg i y [
51
].
Impo an ly, loss o chlo ophyll, inhibi ed g ow h a es [52,53] and bleaching [54,55] ha e
also been epo ed as a esul o apid desicca ion in mosses. Acco dingly, we ha e also
ound se e e bleaching in lichens esponding o con ol e sus o he ea men s.
The absence o ain all in d yland ecosys ems commonly o ces poikilohyd ic o gan-
isms such as Cladonia angi e ina o ely on hyd a ion (and he e o e ac i i y) wi h each
mo ning dew. Those me abolically ac i e pe iods a e also necessa y o ROS-sca enging
enzymes and o he an i-oxida i e molecules o wo k [
56
,
57
], because hei ac i i y is absen
a low hallus wa e con en s [
58
]. The obse ed nega i e impac o educed leng h o
he we , me abolically ac i e pe iods on Cladonia’s i ali y can be he e o e explained by
a cons ained ca bon gain [
59
] and a lack o p o ec ion om ROS and oxida i e s ess.
The abili y o soil lichens o up ake ca bon was likely d ama ically dec eased due o e-
duc ions in hei we day ime pe iod and ne pho osyn hesis. Acco dingly, Maphangwa
e al. [
48
] ound, in an open- op wa ming chambe expe imen , ha he in e ac ion o
clima e wa ming wi h educed p ecipi a ion inc eased desicca ion damage and educed he
du a ion o pho osyn he ic ac i i y. Li e al. [
60
] ecen ly demons a ed ha annual ca bon
ixa ion o lichen-domina ed bioc us was p omo ed in we yea s because soil lichens ha e
a longe we day ime pe iod o he up ake o a ailable ca bon ia pho osyn hesis. In ac ,
bioc us pho osyn hesis seems o be de e mined by he wa e con en a he han ei he
pho osyn he ically ac i e adia ion o empe a u e [60,61].
The lowes F /Fm alues in RW samples we e obse ed in July and Augus , sugges -
ing ha inc eased empe a u es can exace ba e e ec s in samples subjec ed o educed
wa e ing. La son [
62
] documen ed ha p e- ea men wi h inc eased empe a u e we e
able o p oduce a nega i e ca bon balance in lichen species o he genus Umbilica ia and
ha he longe he du a ion o he exposu e, he lowe he empe a u e needed o a ec
ne pho osyn hesis and gas exchange. The coupling o ca bon de ici s wi h high empe a-
u es and cons ained mois u e was plausibly used also o explaining simila esul s in
bioc us s domina ed by mosses [
63
]. Wa ming educed he co e , ichness and e enness
o lichen-domina ed bioc us s in d ylands [
64
]. The e o e, he pho osyn he ic ac i i ies o
lichens and ca bon ixa ion a e expec ed o be sensi i e o wa ming in he sho e m [48].
Ni ogen addi ion imp o ed he i ali y o soil lichens subjec ed o educed wa e ing.
Al hough no signi ican , di e ences we e ound be ween RW and RW+N (p= 0.076),
sugges ing ha N supply inc eased C. angi e ina
´
s i ali y, pa icula ly when acing hea
s ess. As o p e iously s udied e icolous lichens [
65
], C. angi e ina lacks mechanisms o
down- egula e hei N up ake wi h inc easing N load. Mos lichens ha e e ol ed in N-poo
ecosys ems [
66
] and a e he e o e adap ed o low-nu ien en i onmen s and ake up high
amoun s o esou ces whene e a ailable [
67
]. While we expec ed ha ou long- e m
N ea men would ha e a oxic e ec on C. angi e ina, ou indings implied ha he N
addi ion ea men was below he oxici y h eshold, which could be because he ea men
du a ion was insu icien o he N o become oxic, o he concen a ions we e ole able. I
is possible ha he N- e ilized lichens in his expe imen used he abso bed N o inc ease
in es men in i s pho osyn he ic capaci y, as obse ed in o he e iliza ion s udies wi h
epiphy ic lichens [
68
]. Acco dingly, p o eomic analyses p o ed ha Cladonia po en osa
in es ed in p o eins linked o he ene ge ic me abolism in esponse o N s ess [69].
In ag eemen wi h ou second hypo hesis, he long- e m exposu e o N addi ion
con ibu ed o he acclima ion o highe N a ailabili y along he ime. The abili y o cope
wi h long- e m N deposi ions has al eady been obse ed in o he Cladonia species [
19
].
This is in line wi h he hypo hesis ha ole ance o inc eased N may be aligned wi h he
capaci y o main ain balanced ca bon o N s oichiome y among he symbion pa ne s
when high N a es a e supplied h ough he in es men o he ex a N in o mo e pho obion
cells [
70
]. Inc eased ene gy p oduc ion has also been obse ed in Cladonia po en osa o
suppo changes in he mycobion p o eome in esponse o N s ess [
69
]. I is in e es ing o
J. Fungi 2022,8, 333 9 o 13
no e ha he e we e no signi ican di e ences be ween he esponse o Cladonia angi e ina
o 40 and 80 kg N ha
−1
yea
−1
o any ea men , indica ing ha his lichen can acclima e
o la ge a ia ions in en i onmen al N supply be o e eaching he oxici y h eshold.
Many s udies ha e unde lined he complex in e play be ween clima e and ai pollu-
ion [
71
–
74
], he mains eam idea being, o a long ime, ha he lichen mainly esponds o
pollu an s. This wo k indica es ha inc eased a idi y can be a majo c i ical ac o limi ing
lichens’ i ali y, a hypo hesis al eady explo ed by o he au ho s [75–77].
We coupled he physiological esponses obse ed in his expe imen wi h ou al e na-
i e u u e scena ios o global change accoun ing o he in e play o al e a ions in a idi y,
empe a u e, and N deposi ion o o m quali a i e p ojec ions. The ou possible scena -
ios p esen ed in Table 3we e elabo a ed acco ding o he p ojec ions o a la ge numbe
o Global Ci cula ion Models belonging o he Coupled Model In e compa ison P ojec
Phase Six (CMIP6) and N deposi ion global scale e alua ions [
78
] o Medi e anean a eas
loca ed in Eu ope in he nex cen u y. Mos clima e models p ojec an o e all inc eased
a idi y in his a ea ha could be coupled o no wi h inc eased N deposi ion (Scena ios 1
and 2 in Table 3). Based on he esul s om his s udy, bo h scena ios would mode a ely
dec ease C. angi e ina’s i ali y. Scena io 3, accoun ing o inc eases in all he h ee ac o s
conside ed in his s udy (a idi y, N deposi ion and empe a u e), would cause a mo e acu e
de imen al e ec on his lichen’s i ness. E en mo e se e e consequences would be d awn
om Scena io 4, as inc eased a idi y and empe a u e wi hou a aise in N supply unde
he oxici y h eshold would cause a d ama ic educed i ness in C. angi e ina.
Table 3.
P ojec ions o he i ali y o Cladonia and o he lichens wi h simila ecology based on
ou global change scena ios o he nex cen u y. Componen s o each scena io we e ga he ed
om he li e a u e, and p edic ions o lichen i ali y we e made based on ou pu s om his s udy.
A ows poin ing upwa d indica e an inc easing endency, while hose poin ing downwa d ep esen
a dec ease. Mo e a ows indica e a g ea e e ec .
Scena io A idi y Ni ogen
Deposi ion Tempe a u e Lichen Vi ali y
1↑- - ↓
2↑ ↑ -↓
3↑ ↑ ↑ ↓↓
4↑-↑ ↓↓↓
P ojec ions o d yland o ganism esponse o global change emain unde deba e.
Some s udies sugges plan and soil ood webs o hese ecosys ems a e ela i ely esilien
o clima e changes, as hey a e adap ed o ex eme en i onmen s [
79
,
80
]. Ou s udy, along
wi h o he s, sugges s ha many d yland o ganisms a e al eady li ing a he edge o hei
ecological ole ance and could s ongly and non-linea ly espond o e en sub le changes in
clima e [81–84].
5. Conclusions
The wo k p esen ed he e sugges s ha (1) educed wa e ing was a key ac o de-
c easing he i ali y o he soil lichen Cladonia angi e ina; (2) N addi ion imp o ed s ess
ole ance o high empe a u es; and (3) he long- e m exposu e o N addi ion con ibu ed
o he acclima ion o highe N a ailabili y. Despi e con en ional hough ha inc eased N
will a ec (e en linea ly) ecophysiological esponses in soil lichens, his wo k illus a es
ha he ac ual beha io depends on in e ac ions wi h o he d i e s o global change. The
in e ac ions be ween educed wa e ing, inc eased N supply and empe a u e ha may
occu in his egion ha e he po en ial o ad e sely a ec he physiological pe o mance o
his soil lichen. Cladonia angi e ina and, p esumably, o he lichens wi h simila ecology can
cope be e wi h inc eased a idi y i hey bene i om inc eased N supply below he oxici y
h eshold. Dec eased physiological pe o mance o bioc us - o ming o ganisms in d yland
egions may likely al e he complex s uc u e and mul i unc ionali y o Medi e anean
