Ci a ion: Ga cía-Robles, H.; Cañadas,
E.M.; Lo i e, J.; Fe nández-Ondoño, E.
T ade-O be ween Facili a ion and
In e e ence o Allelopa hic
Compounds in Vege a ion Reco e y:
The Case o Rosma inus o icinalis in
Deg aded Gypsum Habi a s. Plan s
2022,11, 459. h ps://doi.o g/
10.3390/plan s11030459
Academic Edi o s: Bousque -
Mélou Anne, James M Mwendwa
and Sajid La i
Recei ed: 19 Janua y 2022
Accep ed: 5 Feb ua y 2022
Published: 7 Feb ua y 2022
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plan s
A icle
T ade-O be ween Facili a ion and In e e ence o Allelopa hic
Compounds in Vege a ion Reco e y: The Case o Rosma inus
o icinalis in Deg aded Gypsum Habi a s
Helena Ga cía-Robles 1,* , E a Ma ía Cañadas 1, Juan Lo i e 1and Emilia Fe nández-Ondoño 2
1Depa amen o de Bo ánica, Uni e sidad de G anada, Campus Fuen enue a s/n, 18071 G anada, Spain;
ecanadas@ug .es (E.M.C.); jlo i e@ug .es (J.L.)
2Depa amen o de Eda ología y Química Ag ícola, Uni e sidad de G anada, Campus Fuen enue a s/n,
18071 G anada, Spain; e e nand@ug .es
*Co espondence: helenag @ug .es
Abs ac :
Rosma inus o icinalis ad an ageously compe es wi h o he species in es o ed gypsum
ou c ops,and u he esea ch is needed o unde s and he causes. Speci ically, we ocus on he
po en ial allelopa hic e ec s de i ed om i s e penes on he eme gence o gypsum species. To his
end, we es ablished 120 ci cula subplo s in a p e iously es o ed gypsum ou c op, and andomly
applied ou di e en ea men s based on he p esence/absence o osema y plan s and hei lea es
on he soil. A e wa ds, we conduc ed an expe imen al sowing o na i e gypsophiles. All subplo s
we e moni o ed o es ima e seedling eme gence, and soil and lea samples we e analysed o e penes.
The esul s show ha he ea men s had signi ican e ec s on he o e all eme gence o seedlings,
and e penes we e ound in osema y lea es and soils, wi h no signi ican di e ences in e pene
composi ion. In pa icula , we iden i ied a clea nega i e e ec in he ea men whe e osema y plan s
we e elimina ed bu i s lea es we e le along wi h allelopa hy (2.57
±
0.54 indi iduals/subplo ).
Unexpec edly, he p esence o osema y plan s seems o acili a e he eme gence o gypsum species
(9.93
±
1.61 indi iduals/subplo ), coun e ac ing he e ec s o he allelopa hic subs ances in he
soil. Consequen ly, we do no sugges emo ing osema y plan s in ea ly s ages o encou age he
eme gence o gypsum species in es o ed a eas.
Keywo ds: mining es o a ion; allelopa hic compounds; essen ial oils; acili a ion
1. In oduc ion
Gypsum habi a s a e sp ead ac oss he wo ld and comp ise app oxima ely 100 million
hec a es [
1
]. Despi e he ac ha hey a e spa sely ep esen ed all o e he wo ld, hei
impo ance is c ucial. Co e ing 35,487 km
2
, he Ibe ian Peninsula boas s he la ges a ea
o gypsum ou c ops in Eu ope [
2
]. They ha bou an excep ionally adap ed lo a, which is
unique wi hin he Eu opean con inen [
3
,
4
]. The peculia edaphic cha ac e is ics o gypsum
habi a s de ine and de e mine he lo a inhabi ing hem. This is he eason why he e
a e so many endemic and na i e species in hese pa icula a eas, he so-called “gypsum
species” as hey shall be he ea e e e ed o as, which can be di ided in o “gypsophiles”
and “gypso ags”. In his con ex , “gypsophiles” ha e been de ined as hose plan species
es ic ed o gypsum soils, and “gypso ags” as hose plan species commonly occu ing on
bo h gypsum and non-gypsum subs a es [5].
Despi e gypsum habi a s being conside ed a p io i y o conse a ion [
6
], la ge gypsum
a eas and, hus, gypsum lo a a e dis u bed by qua ying [
7
–
9
] and ep esen a majo
challenge in he es o a ion o dis u bed singula en i onmen s [
10
]. In pa icula , he
Ibe ian gypsum ege a ion is being se e ely damaged since Spain is one o he main
gypsum p oduce s wo ldwide [
11
]. On his basis, p e ious wo ks ha e demons a ed he
limi a ions ha na u al succession has o he eco e y o gypsum ege a ion o e he sho
Plan s 2022,11, 459. h ps://doi.o g/10.3390/plan s11030459 h ps://www.mdpi.com/jou nal/plan s
Plan s 2022,11, 459 2 o 15
o medium e m, i.e., [
7
,
8
,
12
,
13
]. Consequen ly, he e is a need o de elop speci ic measu es
o es o e hese en i onmen s.
The eco e y o gypsum a eas has been sa is ac o ily app oached h ough di e en
echniques [
14
–
18
]. Pa icula ly, in a o me expe imen , Balles e os e al. [
8
] ound a
balanced eme gence, su i al, and g ow h in di e en subs a e mix u es o he sown
gypsum species (bo h gypsophiles and gypso ags). One o he sown gypso ags in ha
expe imen was osema y (Rosma inus o icinalis L.), which wel e yea s la e p esen ed
a signi ican ly highe co e age in all he ea men s applied compa ed wi h he o he
sown species [
19
]. Rosema y is a Medi e anean na i e species cha ac e ised by a high
ole ance o hea , d ough , and o poo , d y, sandy, and ocky soil ypes [
20
]. F om a
phy ochemical poin o iew, and simila ly o o he species o he Lamiaceae amily [
20
,
21
],
osema y is cha ac e ised by he p esence o essen ial oils wi h allelopa hic p ope ies,
which could explain i s compe i i e success agains o he species. Allelopa hy is he
p ocess in which plan s elease phy o oxic compounds in o he en i onmen o inhibi
he ge mina ion and g ow h o o he plan s sha ing he same habi a [
22
]. Consequen ly,
allelopa hy plays a signi ican ole in plan dominance, succession, species di e si ica ion,
and he es ablishmen o plan communi ies [
23
–
27
]. Wa dle e al. [
28
] sugges ed ha
allelopa hic inhibi ion may be mo e likely o occu in communi ies wi h low species
ichness, such as he gypsum habi a in he s udy a ea, in which soil biochemis y is likely
o be de e mined by dominan plan species. In his sense, osema y essen ial oils ha e
been p o en o inhibi o educe seed ge mina ion (a key s age in plan de elopmen )
o some weeds and c ops such as Silybum ma ianum (L.) Gae n o Raphanus sa i us
L. [
21
,
29
], which is he eason o which his species has been p oposed as a biohe bicide
o con ol seed ge mina ion [
20
,
29
,
30
]. Essen ial oils a e a complex mix u e o e penes
and o he a oma ic and alipha ic compounds in di e en p opo ions [
31
], whose po en ial
syne gic in e ac ions a e no ye well known [
32
]. None heless, allelopa hy is commonly
desc ibed o indi idual e penes in he bibliog aphy [
32
]. Fo ins ance,
α
- and
β
-pinene,
campho , bo neol, ca ac ol, hymol, eucalyp ol (o 1–8 cineole), limonene, o-cymenone,
ci onellol, camphene,
α
-phelland ene, and p-cymene ha e been ex ensi ely desc ibed as
eme gence inhibi o s o e a de s [
20
,
21
,
33
–
35
]. Wi h ega d o he inhibi o y e ec ha
R. o icinalis essen ial oils ha e on he ge mina ion o some plan s such as S. ma ianum and
R. sa i us [
21
,
29
], mono e penes and sesqui e penes [
36
], such as eucalyp ol and bo neol,
a e he main esponsible compounds [
20
,
21
]. Mo eo e , Sc i an i e al. [
37
] epo ed ha
mono e pene ans e o he soil is highe in ho and d y clima es, ac ing as impo an
allelopa hic agen s. Conside ing all he abo e, R. o icinalis could limi he eco e y o
deg aded gypsum habi a s in which ge mina ion is a de e mining s age due o he ha sh
edaphic condi ions [5,10].
Consequen ly, in his s udy, we aimed o explo e he po en ial in e e ence o osema y
in he eme gence o o he gypsum species such as Ononis iden a a L. subsp. c assi olia
(Du ou ex Boiss.) Nyman, Helian hemum squama um (L.) Dum. Cou s and Lepidium
subula um L., ocusing on he ole o i s allelopa hic compounds. Finally, based on he
esul s, we seek o con ibu e o he enhancemen o es o a ion plans o gypsum habi a s,
which a e a p io i y o conse a ion.
2. Resul s
2.1. E ec s o T ea men s on Seedling Eme gence
2.1.1. O e all Eme gence
The o e all eme gence o pe ennial species as a g oup was a he low in he non-
sown expe imen al a eas (bo h wi h and wi hou osema y in luence) and did no show
signi ican di e ences om one ano he (Figu e 1). Meanwhile, he ea men s applied
in he sown subplo s indeed p esen ed signi ican e ec s on he o e all eme gence o
seedlings (Figu e 1and Supplemen a y Table S1). In pa icula , he “Rosma inus o icinalis”
ea men egis e ed he highes o e all eme gence (9.93
±
1.61 indi iduals pe subplo ),
wi h signi ican di e ences compa ed o he o he ea men s. On he o he hand, he
Plan s 2022,11, 459 3 o 15
lowes o e all eme gence appea ed in “Li e all” ea men (2.57
±
0.54 pe subplo ),
which also had signi ican di e ences compa ed o he o he s. E en ually, he “Ba e Soil”
(
4.30 ±0.55 pe subplo
) and “Rosema y Remo al” (5.07
±
0.75 pe subplo ) ea men s
p esen ed an in e media e o e all eme gence and no signi ican di e ences wi h each o he .
Figu e 1.
Mean (
±
SE) eme gence o pe ennial species (n
º
o seedlings pe subplo and ea men ).
T ea men s: RO: Rosma inus o icinalis; LF: Li e all; RR: Rosema y Remo al; BS: Ba e Soil; NS: non-
sown subplo s wi hou osema y; NSR: non-sown subplo s wi h osema y. Di e en le e s ep esen
s a is ically signi ican di e ences (p< 0.05) o he pos hoc Tukey es s pe o med a e he GLMMs,
whe e “a” ep esen s he lowes alue and “c” he highes .
Focusing on he key species as a g oup (Ononis iden a a subsp. c assi olia,Helian he-
mum squama um,Lepidium subula um,Thymus zygis subsp. g acilis,Rosma inus o icinalis,
H. sy iacum, and S ipa enacissima), o on he sown species as a g oup (O. iden a a subsp.
c assi olia,H. squama um and L. subula um), he same pa e n was ound o eme gence
(Figu es 2and 3, espec i ely).
Figu e 2.
Mean (
±
SE) eme gence o key species (n
º
o seedlings pe subplo and ea men ). T ea -
men s: RO: Rosma inus o icinalis; LF: Li e all; RR: Rosema y Remo al; BS: Ba e Soil; NS: non-sown
subplo s wi hou osema y; NSR: non-sown subplo s wi h osema y. Di e en le e s ep esen
s a is ically signi ican di e ences (p< 0.05) o he pos hoc Tukey es s pe o med a e he GLMMs,
whe e “a” ep esen s he lowes alue and “c” he highes .
Plan s 2022,11, 459 4 o 15
Figu e 3.
Mean (
±
SE) eme gence o he sown species (n
º
o seedlings pe subplo and ea men ).
T ea men s: RO: Rosma inus o icinalis; LF: Li e all; RR: Rosema y Remo al; BS: Ba e Soil; NS: non-
sown subplo s wi hou osema y; NSR: non-sown subplo s wi h osema y. Di e en le e s ep esen
s a is ically signi ican di e ences (p< 0.05) o he pos hoc Tukey es s pe o med a e he GLMMs,
whe e “a” ep esen s he lowes alue and “c” he highes .
2.1.2. Eme gence Pe Species
Conside ing all he sown subplo s, he e has been li le eme gence o seedlings o
O. iden a a subsp. c assi olia (14 indi iduals), L. subula um (22 indi iduals), S. enacissima
(3 indi iduals), and H. sy iacum (13 indi iduals), while a much highe eme gence o
H. squama um (373 indi iduals) and T. zygis subsp. g acilis (137 indi iduals) seedlings
occu ed. In be ween, R. o icinalis had an a e age eme gence (52 seedlings).
Despi e he gene ally low eme gence egis e ed o mos o he species, mul iple
compa isons (Tukey’s es ) showed signi ican di e ences be ween ea men s o nea ly all
he key species (Table 1). Mo e p ecisely, he “Ba e Soil” ea men p omo ed he highes
eme gence wi h signi ican di e ences compa ed o he o he ea men s o L. subula um
and H. sy iacum, whe eas “Rosma inus o icinalis” was he mos a ou able ea men o
H. squama um,R. o icinalis and T. zygis subsp. g acilis. In he case o O. iden a a subsp.
c assi olia and S. enacissima, eme gence was equally low in all he ea men s.
Table 1.
Mean (+SE) eme gence (n
º
o seedlings) pe species and ea men . Di e en le e s ep esen
s a is ically signi ican di e ences (p< 0.05) o he pos hoc Tukey es s pe o med a e he GLMMs,
whe e “a” ep esen s he lowes alue and “d” he highes . T ea men s: BS: Ba e Soil; LF: Li e all;
RR: Rosema y Remo al; RO: Rosma inus o icinalis.
Key Species Eme gence (Mean + SE) Pe T ea men
RO LF RR BS
O. iden a a subsp. c assi olia 0.13 ±0.10 a 0.13 ±0.08 a 0.07 ±0.04 a 0.13 ±0.06 a
H. squama um 6.03 ±1.25 d 0.67 ±0.19 a 3.50 ±0.71 c 2.23 ±0.39 b
L. subula um 0.13 ±0.08 ab 0.03 ±0.03 a 0.07 ±0.05 a 0.50 ±0.20 b
R. o icinalis 1.4 ±0.89 b 0.13 ±0.08 a 0.10 ±0.07 a 0.10 ±0.06 a
H. sy iacum 0.17 ±0.11 ab 0.00 ±0.00 a 0.03 ±0.03 a 0.23 ±0.08 b
T. zygis subsp. g acilis 2.03 ±0.79 b 0.97 ±0.29 a 0.53 ±0.24 a 1.03 ±0.20 a
S. enacissima 0.00 ±0.00 a 0.00 ±0.00 a 0.10 ±0.07 a 0.00 ±0.00 a
Plan s 2022,11, 459 5 o 15
2.2. Essen ial Oils in Soils and Lea es
Ch oma og aphic analyses o essen ial oils con i med ha he e we e mono e penes
and sesqui e penes in lea es (GL and FL) and in he soils in luenced by osema y plan s
(RS). Mo e p ecisely, a o al o 39 e penes we e isola ed (Table 2and Supplemen a y
Table S2), 10 o which a e “key e penes” wi h desc ibed allelopa hy [
20
,
21
,
33
–
35
]. On he
con a y, he e we e no e penes in he ba e soils (BS) (Table 2and
Supplemen a y Table S2
).
Table 2.
P esence o e pene compounds iden i ied by GC–MS in he essen ial oil o R. o icinalis lea
and soil samples. “X” means “p esence o e pene”. GL: g een lea es; FL: allen lea es; BS: ba e soil;
RS: soil unde osema y plan .
NTe pene Compound Re en ion Time
(Seconds)
Key
Te pene
Sample Types
Lea es Soils
GL FL BS RS
1 icyclene 7.34 No X X
2 hujene 7.46 No X
3α-pinene 7.59–7.78 Yes X X X
4camphene 8.11–8.27 Yes X X X
5 e benene 8.22 No X
6β-pinene 9.03–9.19 Yes X X X
71-oc en-3-ol 9.21–9.27 No X
8 3- oc anone 9.34–9.45 No X
9β-my cene 9.51–9.65 No X X X
10 3- hep anol 9.86 No X
11 α-phelland ene 10.07–10.18 Yes X X
12 a emisa iene 10.45–10.56 No X
13 p-cymene 10.74–10.84 Yes X X X
14 limonene 10.94–11.05 Yes X X X
15 eucalyp ol (1’8 cineole) 11.04–11.16 Yes X X X
16 7_ e pinene 11.97–12.06 No X X
17 e pinolene 12.97–13.04 No X
18 linalool 13.51–13.61 No X X X
19 3.5 hep adien-2-ol-2.6 14.26–14.33 No X X X
20 camphenol 14.45–14.53 No X
21 pinoca eol 14.99–15.13 No X
22 campho 15.17–15.36 Yes X X X
23 iso-pinocamphone 15.66–15.73 No X
24 bo neol 16.10–16.21 Yes X X X
25 e pinen-4-ol 16.41–16.49 No X X X
26 α- e pineol 16.97–17.02 No X X X
27 α-san olin-alcohol 17.30 No X
28 e benone 17.42 No X X X
29 bo nyl ace a e 20.04 No X
30 ca ac ol 20.3 Yes X
31 copaene 23.11 No X
32 ca yophyllene 24.52–24.61 No X X X
33 a omadend ene 25.63 No X X
34 cis-α-bisabolene 25.715 No X X X
35
me hyl 8
0
11
0
14
0
17-eicosa e aenoa e
27.544 No X
36 ca yophyllene oxide 29.42 No X X X
37 a nesol 29.51–30.00 No X
38 idecan 31.05 No X
39 ledene oxyde 31.58 No X
Plan s 2022,11, 459 6 o 15
The essen ial oil o g een lea es p esen ed a leas 24 e penes, and he e we e 23
in he allen lea es, sha ing a o al o 18 di e en e penes (Table 2). In he case o soil
samples, he e we e no e penes in he “Ba e Soil”, whe eas we ound a leas 30 di e en
e pene compounds in he soils unde osema y plan s (RS), among which 20 we e also
ound in lea samples (Table 2). Acco ding o he a ea (%) occupied by each e pene in
he ch oma og aphic cu es (Supplemen a y Table S3), he mos abundan e penes in
g een lea es (mo e han 90% o he o al) a e
α
- and
β
-pinene, camphene,
β
-my cene,
limonene, eucalyp ol,
α
- e pinene, e pinolene, campho , bo neol, bo nyl ace a e and
ca yophyllene, among which se en a e “key e penes”. In he case o allen lea es, he mos
abundan e penes (app oxima ely 80–90% o he o al) a e
α
-pinene, camphene, p-cymene,
eucalyp ol, campho , bo neol,
α
- e pineol, ca yophyllene, among which six o hem a e
“key e penes”. Finally, he mos abundan e penes in osema y soil samples a e
α
- and
β
-pinene, camphene, eucalyp ol, campho , bo neol, e benone, and ca yophyllene, among
which six a e “key e penes”.
Mul iple compa isons a e a mul i a ia e analysis o he o e all e pene con en
(Table 3) show signi ican di e ences be ween g een lea es (GL) and allen lea es (FL) and
be ween g een lea es (GL) and soils unde osema y plan s (RS). None heless, he e a e
no signi ican di e ences be ween he e pene con en in allen lea es (FL) and soils unde
osema y plan s (RS).
Table 3.
Signi ican di e ences (p< 0.05) be ween sample ypes o he pos hoc es pe o med a e
mul i a ia e analysis. Signi ican codes: ‘*’ 0.05. GL: g een lea es; FL: allen lea es; RS: soil unde
osema y plan .
Pai s D SumsO Sqs F.Model R2 p. Value p. Adjus ed Sig
FL s. GL 1.00 0.27 4.92 0.38 0.01 0.02 *
FL s. RS 1.00 0.13 1.17 0.13 0.32 0.97
GL s. RS 1.00 0.33 3.55 0.31 0.01 0.02 *
Mul iple compa isons be ween sample ypes pe key e penes a e pe mu a ion es s
o linea models a e shown in Table 4. To highligh , signi ican di e ences we e ound
be ween “BS” samples and he es o he sample ypes o all key e penes excep o
α
-phelland ene and ca ac ol, o which he e we e no signi ican di e ences be ween
any sample ypes. In he case o p-cymene and limonene, he e a e ma ginal signi ican
di e ences among “GL”, “FL”, and “RS”, and o
β
-pinene, he e a e signi ican di e ences
be ween “GL” and “FL”.
Table 4.
Signi ican di e ences be ween he sample ypes pe key e pene. Di e en le e s ep esen
s a is ically signi ican di e ences (p< 0.05) o he pos hoc es pe o med a e lmPe m, whe e “a”
ep esen s he lowes alue and “c” he highes . GL: g een lea es; FL: allen lea es; RS: soil unde
osema y plan ; BS: ba e soil.
Te penes
Sample Types
Lea es Soils
GL FL BS RS
α-pinene b b ab
camphene b b ab
β-pinene c ab abc
α-phelland ene a a a a
p-cymene ab b aab
limonene b ab aab
eucalyp ol b b ab
campho b b ab
bo neol b b ab
ca ac ol a a a a
Plan s 2022,11, 459 7 o 15
3. Discussion
Resul s e ealed clea signi ican e ec s o ea men s on he gene al eme gence o
gypsum species (bo h gypsophiles and gypso ags), con i ming he hypo hesis ha ose-
ma y condi ions he eme gence o seedlings in gypsum a eas (Table 5). In pa icula ,
we obse ed a clea nega i e e ec o he “Li e all” (LF) ea men , in which osema y
plan s we e elimina ed bu he lea es emained, which could be linked o he p esence o
allelopa hic compounds.
Table 5.
Summa y o he main cha ac e is ics o ea men s and hei e ec o e eme gence.
“+” = posi i e e ec
; “
−
“ = nega i e e ec . T ea men s: RO: Rosma inus o icinalis; RR: Rosema y
Remo al; LF: Li e all; BS: Ba e Soil.
T ea men s
Rosema y
Plan
P esence
Rosema y
Lea es
P esence
Allelopa hic
Compounds
Facili a ion
E ec
E ec o e
Eme gence
RO Yes Yes Yes Yes +++
LF Remo al Yes Yes No –
RR Remo al Remo al Residual No +
BS Ne e Ne e No No +
Acco ding o he ch oma og aphical analyses conduc ed on lea and soil essen ial oils,
he e a e ou clea pa e ns ega ding e pene composi ion: (1) osema y- ee soils a ely
con ain e penes; (2) lea samples, bo h g een and allen, con ain e penes wi h simila
esul s o hose ound by O meño e al. [
38
] and Alipou e al. [
39
]; (3) soils somehow
in luenced by osema y plan s also con ain e penes; and (4) he samples o osema y allen
lea es, osema y g een lea es and osema y soils p esen ed simila e pene composi ions,
cha ac e ised by he p esence o e penes p e iously desc ibed as eme gence inhibi o s o
e a de s, such as
α
- and
β
-pinene, camphene, p-cymene, limonene, eucalyp ol, campho
and bo neol [
20
,
21
,
33
–
35
]. Consequen ly, ou esul s e eal he e is a s eam o e penes
om osema y plan s and lea es o he nea by soils, simila ly o wha Fishe [
33
] obse ed,
sugges ing a co ela ion be ween e pene p esence and he inhibi ion o seedling eme gence,
especially o O. iden a a subsp. c assi olia, L. subula um, H. sy iacum, and S. enacissima.
Mo eo e , as a esul o such e pene ans e , he sown subplo s somehow in luenced by
osema y may ha e simila e pene composi ions, including hose ha ing unde gone he
“Rosema y Remo al” ea men , in which e penes may emain o a pe iod o ime a e
osema y plan and allen lea es emo al [40].
In con as o wha could be expec ed, “Rosma inus o icinalis” ea men had a signi i-
can posi i e e ec on he gene al eme gence o pe ennial species despi e i s po en ially
high load o allelopa hic e penes. Fu he mo e, he highes eme gence was ound o
his ea men , which made us conside he acili a ion p ocess as a de e mining ac o
in he ea ly s ages o plan eme gence in s ess ul en i onmen s, mo e han allelopa hy
could be [
41
]. Rega dless o hei ole as allelopa hic compound p oduce s, adul osema y
plan s could o e shade and o he bene i s o he species eme ging nea by (i.e., a sa e
egene a ion o he mic o-niche, highe wa e and nu ien a ailabili y, e c.), which has been
p o en o be o high ele ance in deg aded Medi e anean a eas [
42
]. Ac ually, Pugnai e,
and Luque [
43
] indica ed ha a combina ion o compe i ion and acili a ion e ec s o en
ope a es simul aneously among plan species in na u e.
In e es ingly, he lowes gene al eme gence was egis e ed o he “Li e all” ea men .
When osema y plan s we e emo ed along wi h hei acili a ion e ec , bu osema y
allen lea es emained, a s ong inhibi o y e ec o e he eme gence o he key species was
obse ed, p obably due o he high con en o allelopa hic e penes. To mo-Blanes e al. [
44
]
also de ec ed ha osema y allen lea es had nega i e e ec s on he eme gence o pe ennial
species when sowing was conduc ed on he li e laye . Scognamiglio e al. [
27
] ound ha
he deg ada ion o allen lea es by mic oo ganisms eleases allelopa hic subs ances, such as
Plan s 2022,11, 459 8 o 15
e pene compounds, which inhibi s ge mina ion and/o plan de elopmen . In addi ion o
his, Alias-Gallego e al. [
40
] epo ed ha unde a id condi ions, allelopa hic compounds
a e mainly eleased in o he soil h ough he deg ada ion o li e .
Rosema y- ee soils, hose wi h “Ba e Soil” ea men , which a e p esumably d ie ,
mo e compac ed, nu ien -poo , osema y e penes- ee, and ecei e di ec insola ion,
showed a simila end compa ed o soils ha ing unde gone he “Rosema y Remo al”
ea men , in which he osema y plan s and allen lea es we e emo ed o he expe imen .
Bo h p esen ed lowe eme gence a es han he “Rosma inus o icinalis” ea men , bu highe
han hose o he “Li e all” ea men . The ac ha eme gence in he “Ba e Soil” and
“Rosema y Remo al” ea men s was usually compa able sugges ed ha , i he sou ce o
e pene p oduc ion (ei he osema y plan s o allen lea es) was emo ed, mono e penes
( ola ile ac ion o essen ial oils) and sesqui e penes would e apo a e and decompose
wi h ime [45] and hus, he inhibi ing e ec owa d o he plan species would hen cease.
Focusing on he sown species, he e was li le seedling eme gence o O. iden a a subsp.
c assi olia and L. subula um in he expe imen al a ea, whe eas a much highe eme gence o
H. squama um occu ed. The eme gence o O. iden a a subsp. c assi olia was e y low in all
he ea men s p obably due o i s commonly low ge mina ion a e (
≤
20%)
[46,47]
, oge he
wi h he low a io o sown seeds o his species compa ed o he o he s. L. subula um
p esen ed an especially low eme gence a e in hose ea men s somehow in luenced by
a osema y plan , whe eas i ound i s peak o eme gence in he “Ba e Soil” ea men ,
sugges ing ha his species is especially sensi i e o compe i ion and allelopa hy in e e -
ence. Escude o e al. [
48
] obse ed ha he seedlings o L. subula um end o es ablish on
he gypsum su ace c us and limi hei habi a o a educed niche beyond he scope o
compe i i e in e ac ion wi h o he sh ub species. Mo eo e , Escude o e al. [
49
] obse ed
how he eme gence o gypsophiles in gypsum soils could ha e been nega i ely a ec ed by
he allelopa hic e ec s o some gypso ags. On he con a y, H. squama um is able o g ow
in a ield wi h a wide a ie y o soils, al hough i s su i al a e and g ow h a e highe on
genuine gypsum soils [
50
]. No ably, H. squama um was he species which bene i ed he
mos om e e y ea men , especially om “Rosma inus o icinalis”. Fo onda e al. [
51
] also
obse ed he abundance o H. squama um seedlings unde he in luence o a nu se species
and in open a eas. In addi ion o his, H. squama um seems o be a s ong compe i o in
gypsum habi a s, since in he cases in which he eme gence o his species ollowed an
explosi e end, no seedlings o o he species (o e y ew, as in “Rosma inus o icinalis”
ea men ) accompanied hem, p obably due o he sca ci y o esou ces.
In he case o gypso ags, he esponse o seedling eme gence o ea men s was
di e en o each species. I was gene ally low, excep o T. zygis subsp. g acilis. Fo
ins ance, S. enacissima only eme ged in “Rosema y Remo al” ea men , which sugges s
ha his species could ha e been nega i ely a ec ed by shadowing (“Rosma inus o icinalis”
and “Li e all” ea men s), as epo ed by Ga cía-Fayos and Gasque [
52
], and/o by
physical ba ie s such as soil c us (“Ba e Soil” ea men ) [
44
]; mo eo e , i s ex emely
low eme gence in he “Rosema y Remo al” ea men made us hink ha compe i ion
wi h H. squama um and/o osema y-induced allelopa hy could ha e also a ec ed his
species. Al hough H. sy iacum and H. squama um usually coexis in gypsum habi a s [
53
],
a e a pe iod o se e e d ough , bo h popula ions may ha e been desynch onised [
53
],
and H. sy iacum, as a gypso ag species, may ha e been displaced by H. squama um, which
has p o ed o be a s ong compe i o unde hese ci cums ances. Despi e i s o e all low
eme gence, H. sy iacum seemed o be mo e a ou ed ei he by ba e soils whe e he e
is no compe i ion o esou ces, o by soils wi h osema y plan s, possibly due o i s
ole as a nu se species. A e H. squama um,T. zygis subsp. g acilis was he key species
wi h he bes pe o mance. This species eme ged simila ly in all he ea men s excep
in “Rosma inus o icinalis”, whe e we ound i s highes eme gence. T. zygis subsp. g acilis
also p oduces essen ial oils wi h allelopa hic e ec s [
54
] and should ha e he abili y o
selec i ely de ec and espond o chemical signals [
55
]. Mo eo e , T. zygis subsp. g acilis
seems o cope wi h compe i ion e y well [
56
], and as a gypso ag species eme ging in
Plan s 2022,11, 459 9 o 15
gypsum habi a s, i may ha e bene i ed om osema y-induced acili a ion [
51
]. Finally,
R. o icinalis has been obse ed o ac as a di e si y epellen [
51
]. In his sense, allelopa hy
could be a mechanism used by some gypso ags o a oid compe i ion o esou ces and
succeed in gypsum habi a s [
51
]. Despi e he g ea abundance o R. o icinalis adul plan s
in he expe imen al a ea, seedling eme gence o his species was a he low in ea men s,
excep in “Rosma inus o icinalis”, whe e seed ec ui men was e ec i e, p obably due o
seed a ailabili y and acili a ion p ocesses. On op o his, he e was one case in which
osema y seedlings eme ged explosi ely in “Rosma inus o icinalis” ea men , hinde ing
he eme gence o o he species’ seedlings, possibly as a esul o he in e dependence
o esou ce compe ence and allelopa hy [
57
]. In e es ingly, in he “Rosema y Remo al”
and “Li e all” ea men s, eme gence was e y low e en i hese soils should con ain a
signi ican seed bank. Unde hese condi ions, osema y seedlings may ha e su e ed om
au o oxici y, as has been desc ibed o o he allelopa hic species [51,58].
O e all, es o a ion plans o deg aded gypsum habi a s should os e he de elop-
men o species such as O. iden a a subsp. c assi olia, which could ac as a nu se plan
unde hese ci cums ances, as desc ibed o o he species o he Fabaceae amily [
51
], and
hus help s uc u e gypsum communi y. Mo eo e , es o a ion plans should a oid using
allelopa hic species [59].
4. Ma e ials and Me hods
4.1. S udy A ea
This s udy was conduc ed a a gypsum ou c op a ea in Escúza , loca ed in he sou h-
wes e n a ea o G anada (Spain), 37
◦
2
0
N, 3
◦
45
0
W, a 950 m a.s.l., in he icini y o an ac i e
gypsum qua y. The a ea is cha ac e ised by a con inen al Medi e anean clima e, wi h
mode a ely cold win e s, ho summe s, and a 4-mon h d y pe iod wi h wa e de ici . The
annual a e age empe a u e is 15
◦
C; he cooles a e age empe a u e, 7.6
◦
C, is eached in
Janua y, while he ho es a e age empe a u e, 24.2
◦
C, is eached in Augus . The a e age
annual p ecipi a ion is 421.1 mm, mainly occu ing in win e [8].
The s udy a ea is loca ed in he Neogene-sedimen a y basin o G anada. The dominan
subs a e comp ises lime and gypsum combined wi h ma l, which da es om he la e
Miocene [60]. The p e ailing soils in hese ou c ops a e Lep ic gypsisols [61].
The ege a ion is p edominan ly o med o a mosaic o sca e ed pa ches o na u al
plan s g owing in gypsum ou c ops, su ounded by ce eal c ops, oli e g o es o almond
ya ds. Mo e p ecisely, he a ge habi a o his s udy, he gypsum habi a , is included
in he Eu opean Habi a Di ec i e (92/43/EEC) as 1520, “Ibe ian gypsum ege a ion,
Gypsophile alia” [
6
], and is cha ac e ised by h ee exclusi e species o gypsum ou c ops
(gypsophiles), one o which is a local endemism, Ononis iden a a subsp. c assi olia, and
he o he wo, Helian hemum squama um and Lepidium subula um, a e widesp ead in he
gypsum ou c ops o he Ibe ian Peninsula [
8
]. In addi ion, he e a e also o he equen non-
exclusi e species o gypsum ou c ops (gypso ags) such as S ipa enacissima L., Helian hemum
sy iacum (Jacq.) Dum.Cou s., Helian hemum iolaceum (Ca .) Pau., Thymus zygis L. subsp.
g acilis (Boiss.) R.Mo ales, Teuc ium capi a um L. subsp. g acillimum (Rouy) Valdés Be m.
And Sánchez C espo, Rosma inus o icinalis, Hippoc epis bou gaei (Nyman) He ie , and
Fumana hymi olia (L.) Spach Ex Webb [62].
Ou expe imen al si e was se on an old ce eal ield composed o ma ls, and whe e, in
2009, pos -qua ying condi ions we e ec ea ed o p e ious expe imen s [
8
] in 20 plo s o
5 m ×5 m
, using gypsum spoil ( ocky was e a e gypsum mining; see i s physicochemical
cha ac e isa ion in Supplemen a y Table S4) as he bedding ma e ial. A e wa ds, seeds
o se en gypsum species we e sown he e in No embe 2009, including bo h gypsophiles
(O. iden a a subsp. c assi olia, H. squama um, and L. subula um) and gypso ags (S. enacis-
sima,H. sy iacum,T. zygis subsp. g acilis, and R. o icinalis), he so-called “key species” in
his s udy. As o expe imen al plo s o his in es iga ion, we selec ed hose plo s which
cu en ly p esen compa able soil condi ions, such as hose wi h he “sowing” (S) and
“sowing plus o ganic ma e addi ion” (SO) ea men s (5 plo s each) [8].
