Ecophysiological and biochemical responses to cold and heat waves of native Spartina maritima, alien S. densifora and their reciprocal hybrids

Vol.:(0123456789)
Plan a (2025) 261:99
h ps://doi.o g/10.1007/s00425-025-04675-4
ORIGINAL ARTICLE
Ecophysiological andbiochemical esponses ocold andhea wa es
o na i e Spa ina ma i ima, alien S. densi lo a and hei ecip ocal
hyb ids
Rosa ioÁl a ez1· Sal ado A.Fe nandez‑Gonzalez1· Ad iánPe e a‑Bonaño1· Al onsoDeCi es1· JesúsM.Cas illo1·
BlancaGallego‑Té a 1
Recei ed: 14 Decembe 2024 / Accep ed: 15 Ma ch 2025 / Published online: 1 Ap il 2025
© The Au ho (s) 2025
Abs ac
Main conclusion Spa ina hyb ids ou pe o m pa en al species, showing ansg essi e acclima ion o ex eme cli-
ma es. Na i e S. ma i ima demons a es high seasonal adap abili y and in asi e S. densi lo a low physiological impac ,
sugges ing esilience unde clima e change.
Abs ac Ex eme clima ic e en s, such as cold and hea wa es, a e becoming mo e equen , in ense, and p olonged due
o clima e change. Simul aneously, in asi e alien plan species a e al e ing he composi ion o plan communi ies. Bo h
clima e change and he in oduc ion o alien species pose signi ican h ea s o biodi e si y. We s udied he esponses o 25
biochemical and physiological unc ional ai s o na i e Spa ina ma i ima, alien in asi e S. densi lo a and hei ecip ocal
hyb ids o changing en i onmen al condi ions du ing a cold snap in win e and a hea wa e in summe in Guadiana Ma shes
(Sou hwes Ibe ian Peninsula). These ou closely ela ed axa esponded di e en ly o seasonal en i onmen al luc ua ions.
Bo h hyb id axa, pa icula ly S. ma i ima × densi lo a, exhibi ed ansg essi e esponses, allowing hem o display a wide
ange o acclima ion esponses o ai empe a u e compa ed o hei pa en al species. Na i e S. ma i ima also demons a ed a
ela i ely high acclima ion capaci y o seasonal me eo ological changes. In con as , alien S. densi lo a p esen ed ew acclima-
ion esponses o seasonal en i onmen al changes, esponding p ima ily o sedimen salini y a he han o ai empe a u e.
E en so, all ou s udied Spa ina axa appea o be well-adap ed o he occu ence o cold and hea wa es in he Gul o
Cadiz. These indings unde sco e he complexi y o plan acclima ion s a egies in esponse o ex eme clima ic e en s and
highligh he po en ial o hyb id axa o ace he u u e dynamics o sal ma shes unde clima e change.
Keywo ds An hocyanins· Clima e change· Co dg ass· Hyb idiza ion· Medi e anean clima e· The mal s ess
Abb e ia ions
EC Elec ical conduc i i y
Eh Redox po en ial
F0, Fm, F Ini ial, maximal, and a iable luo escence
F /Fm Maximum quan um e iciency o PSII
MDA Malondialdehyde
NPQ Non-pho ochemical quenching
WC Wa e con en
Vmax Maximal a e o pho osyn he ic oxygen
e olu ion
ΦPSII E ec i e quan um e iciency o PSII
In oduc ion
Ex eme clima ic e en s, such as cold and hea wa es,
a e inc easing in equency, in ensi y and du a ion in he
p esen scena io o clima e change (IPCC 2022). These
clima ic changes a e especially ele an o he Medi e -
anean Basin which has been iden i ied as a clima e change
ho spo (No o e al. 2023). Cold and hea wa es expose
plan s o high le els o he mal s ess ha can diminish
pho osyn hesis, a highly sensi i e p ocess o empe a-
u e changes (Cho ancek e al. 2019; G ossman 2023).
Communica ed by Do o hea Ba els.
* Blanca Gallego-Té a
[email p o ec ed]
1 Depa amen o de Biología Vege al y Ecología, Uni e sidad
de Se illa, Ap 1095, 41080Se ille, Spain
Plan a (2025) 261:9999 Page 2 o 17
The mal s ess may educe he e iciency o pho osyn he ic
elec on anspo in bo h pho osys ems and al e pigmen
accumula ion and chlo ophyll (Chl) luo escence quench-
ings (Teskey e al. 2015; Nie ola e al. 2017). Diminished
pho osyn hesis as a esul o he mal s ess can educe
biomass p oduc ion and he capaci y o ace en i onmen al
s esses ha , in u n, may cause b oad biogeog aphic shi s
and egional changes in dis ibu ion (Aagesen e al. 2016;
Liancou e al. 2020).
Toge he wi h clima e change, in asi e alien plan species
a e changing species composi ion o plan communi ies and
cons i u e also a se e e h ea o biodi e si y (F ied e al.
2014; an Kleunen e al. 2018). Some alien plan species can
su e om non- e e sible pho odamage and educed pho-
osyn hesis due o he mal s ess (Zhu e al. 2019). In con-
as , many alien species may exhibi enhanced esponses,
a biochemical and pho ochemical le els, o he mal s ess
han hei na i e congene s (Dua e e al. 2016; Kune
e al. 2022). The physiological challenges posed by clima e
change o na i e and alien plan species can be aced by
pheno ypic plas ici y and local adap a ion. In gene al, in a-
si e species p esen highe pheno ypic plas ici y in hei
pho osyn hesis pe o mance han non-in asi e species
(Da idson e al. 2011). Mo eo e , some alien plan species
ha e adap ed a wide ange o biochemical and physiological
ai s o he clima e in hei in oduced ange, acili a ing
ex ensi e in asions (Fenollosa and Munne-Bosch 2019). In
his con ex , apid global wa ming has enabled alien species
o expand in o egions in which hey p e iously could no
su i e and ep oduce (Wal he e al. 2009; Ka hi esan and
Gualbe 2016). Liu e al. (2017) ound ha ele a ed em-
pe a u e and CO2 en ichmen a ou ed in asi e alien plan s
mo e s ongly han na i e plan s. Ne e heless, in gene al,
bo h in asi e alien plan s and hei na i e coun e pa s usu-
ally unde go simila adap i e e olu ion esponses o physio-
logical pe o mance o global wa ming (Gianoli e al. 2021).
Alien plan species can hyb idize wi h na i e species
and he upcoming hyb ids can show inc eased i ness in
esponses o di e en en i onmen al s esses due o bio-
chemical and physiological ansg essi e ai s (Gallego-
Té a e al. 2019a, 2020a, b; Zhang e al. 2020). In his
con ex , Sun e al. (2015) ound ha hyb idiza ion sligh ly
ca alyzed he ole ance o a hyb id be ween na i e and alien
Sphagne icola species o low empe a u e and weak ligh
condi ions, eco ded as changes in pho osyn he ic and an i-
oxidan unc ional ai s. Ma e nal e ec s also play a el-
e an ole in he unc ional ai exp ession o in e speci ic
hyb ids acing en i onmen al s ess (Filipe and Mon esinos
2016), including he exp ession o pho osyn he ic ai s (Ji
and Jiao 1999). Mo eo e , he o ma ion o hese hyb ids
can be modula ed by inc easing empe a u es in he p esen
scena io o global wa ming (Gallego-Té a e al. 2019b).
The e o e, hyb idiza ions be ween na i e and alien species
a e o g ea conce n o e olu iona y biologis s and ecologis s
in e es ed in quick e olu iona y p ocesses.
Unde s anding he biochemical and physiological
esponses o na i e and alien plan congene species and
hei hyb ids o seasonal me eo ological changes and
ex eme clima ic e en s is c ucial o p edic ing how hese
axa migh cope wi h u u e en i onmen s in he con ex o
clima e change. To ou knowledge, none s udy has analyzed
he biochemical and physiological esponses o na i e and
in asi e congene s and hei hyb ids o cold and hea wa es
unde ield condi ions. Field s udies a e especially impo an
since hey cap u e he in ica e in e play o en i onmen al
ac o s in luencing plan pe o mance and esilience. Fo
example, plan g ow h-p omo ing bac e ia ound in na u al
condi ions can imp o e he ole ance o he pho osyn he ic
appa a us o he mal s ess (Dua e e al. 2023).
Ou s udy sys em was he na i e co dg ass Spa ina ma -
i ima (Cu is) Fe nald (2n = 60), he alien in asi e Spa ina
densi lo a B ongn. (2n = 70) and hei ecip ocal hyb ids
g owing in Guadiana Ma shes (Gul o Cadiz, sou hwes
Ibe ian Peninsula). Spa ina ma i ima is a ounda ion spe-
cies and a ma sh-building halophy e ha domina es Ibe ian
and o he Eu opean and Sou h A ican low sal ma sh zones
(Cas ellanos e al. 1994). Alien Spa ina densi lo a is a neo-
phy e wi h high pheno ypic plas ici y (Cas illo e al. 2018)
ha is in ading con as ing habi a s along he idal g adien
in he Gul o Cadiz (Nie a e al. 2001). The hyb ids S.
ma i ima × densi lo a (2n = c. 95) and S. densi lo a × ma -
i ima (2n = 65) a e s e ile hyb ids ha show some ansg es-
si e ai s in he ield (eg. alle shoo s and highe g ow h
a es han he pa en al species) (Cas illo e al. 2010) and in
esponse o sal s ess unde con olled condi ions (Gallego-
Té a e al. 2018a, 2019a). We s udied he esponses o 23
biochemical and physiological unc ional ai s o ou ou
ocal Spa ina axa o changing en i onmen al condi ions
du ing a cold snap in win e and a hea wa e in summe . We
hypo hesized ha bo h pa en al species would show high ol-
e ance o changing en i onmen al condi ions h ough accli-
ma ion o di e en unc ional ai s and ha bo h hyb ids
would p esen ansg essi e ai s enabling hem o show
highe acclima ion capaci y han pa en al species.
Ma e ials andme hods
S udy a ea
Ou s udy was ca ied ou in he San B uno Ma shes
(37º10ʹ−37º16ʹN, 7º28ʹ−7º16ʹW), loca ed in he sou hwes
o he Ibe ian Peninsula, in he Guadiana Ri e Ma shes
(Fig.S1). The Guadiana Es ua y is a meso idal open es ua y
wi h semidiu nal ides, a mean ange o 2.10m and a mean
sp ing idal ange o 2.97m (Cas ellanos e al. 1994). The
Plan a (2025) 261:99 Page 3 o 17 99
s udy a ea p esen s a gen le slope along a wide idal g adi-
en , showing a clea plan zona ion pa e n wi h low ma shes
domina ed by Spa ina ma i ima and Sa coco nia pe ennis
(Mill.) A.J. Sco , middle ma shes by Halimione po ula-
coides (L.) Aellen, Sa coco nia u icosa (L.) A.J. Sco and
exo ic S. densi lo a, and high ma shes by A h ocnemum
mac os achyum (Mo ic.) C. Koch and Limonias um mon-
ope alum (L.) Boiss. (Gallego-Té a e al. 2018b). The s udy
a ea has a Medi e anean clima e wi h A lan ic in luence
wi h mild and ela i ely we win e s (mean ai empe a u e
is ca. + 11 ºC in Janua y; a e age annual p ecipi a ion is c.
506mm), and ho and d y summe s (mean ai empe a u e
is + 25 ºC in Augus wi h almos no ain all). The s udied
Medi e anean sal ma shes a e exposed o b oad daily and
seasonal changes in empe a u e (> 30°C) and e y high
empe a u es du ing summe ime (> + 40°C) (Boughalleb
e al. 2022). Sedimen salini y le els peak nea seawa e
concen a ion du ing summe d ough s (Con e as-C uzado
e al. 2017). Addi ionally, he s udied Medi e anean sal
ma shes a e exposed o sea le el ise (+ 4.02mm y −1 and
accele a ing) and educed ain alls due o clima e change
(Ko a s e al. 2014; NOAA 2018).
En i onmen al ma ix
Me eo ological da a (minimum, a e age and maximum ai
empe a u e, ºC) o he sampling days and p e ious 9days
in win e and summe we e collec ed om Ayamon e
me eo ological s a ion, loca ed 2km away om he s ud-
ied ma shes (code 4549Y; AEMET 2024). The sedimen-
a y en i onmen was cha ac e ized a he same ime ha
lea samples we e collec ed o labo a o y analyses. We
eco ded sedimen edox po en ial (Eh; mV), pH, elec ical
conduc i i y (EC; mS cm−1) and wa e con en (WC; %) in
he oo zone, be ween 0 and 10cm deep, o e e y ma ked
Spa ina clump (n = 10 samples pe axon). Eh was eco ded
insi u using an elec ode sys em (C ison Ins umen s pH/
mV p-506, Hach Lange Spain, S.L.U., Ba celona, Spain)
(Cas illo e al. 2000). Sedimen s (250ml) we e sampled in
sealed plas ic con aine s and anspo ed o he labo a o y.
Sedimen po e-wa e pH and EC we e eco ded in he un il-
e ed supe na an a e adding dis illed wa e o he sedimen
samples (1:1, / ) using a pHme e (pH/ edox C ison PH25
wi h he p obe C ison 5052) and a conduc i i y me e (C i-
son CM35), espec i ely, in he labo a o y. Sedimen WC
was eco ded by weigh ing ca. 100g o sedimen s be o e
and a e d ying o cons an weigh a + 80 ºC in a o ced-ai
s o e (Cu ado e al. 2014).
Plan ma e ial
Field measu emen s and sample collec ions ook place du -
ing low idal le el a ound sola noon on sunny days and
low ide condi ions in Janua y 2021 du ing a cold snap
associa ed wi h S o m Filomena and in July 2021 du ing
a hea wa e (de ined by he Spanish Me eo ological S a e
Agency (AEMET; ac onym in Spanish) as ≥ 3days wi h
empe a u es abo e he 95 h pe cen ile o July–Augus
1971–2000). All measu emen s we e ca ied ou on he
i s o ally expanded adul lea om adul shoo s o en
andomly selec ed clumps o each axon: na i e Spa ina
ma i ima, alien in asi e S. densi lo a and hei ecip ocal
hyb ids S. ma i ima × densi lo a and S. densi lo a × ma -
i ima. Each axon was sampled along he idal g adien
he e whe e i was mo e abundan . Spa ina ma i ima and
S. ma i ima × densi lo a we e sampled in low ma shes, and
S. densi lo a and S. densi lo a × ma i ima in middle ma shes.
Sampled clumps we e sepa a ed a leas 2m om each o he
o ensu e hey we e di e en indi idual plan s ha we e pe -
manen ly ma ked.
C ude ex ac o labo a o y analyses
Fi s , o ally de eloped adul lea es ( lag lea es) we e col-
lec ed om 5 andomly selec ed plan s om he 10 ma ked
plan o each axa (n = 5 samples pe axon), immedia ely
s o ed in zip-lock plas ic bags wi h silica gel in he ield
and s o ed a −20 ºC once in he labo a o y. To ob ain he
c ude ex ac , 0.2g o lea es we e weighed and p ocessed
in mo a du ing 10–15min wi h 10ml o pu e me hanol
(using sand o help ex ac ion). To elimina e any solid esi-
due, samples we e cen i uged a 9981g, 15min a 10 ºC.
Supe na an was used as a c ude ex ac o he de e mina-
ions o pigmen s, o al an ioxidan capaci y, malondialde-
hyde (MDA) and polyphenol concen a ions.
Pigmen s de e mina ion
The concen a ions o pho osyn he ic pigmen s and an hocy-
anins a e modi ied in esponse o he mal s ess (Ve oshkina
e al. 2023). To assess chlo ophylls (Chl) and ca o enoids
(Ca ) concen a ions om he c ude ex ac (p e iously
dilu ed 10 imes in pu e me hanol), abso bance (Abs) a
665.2nm, 652.4nm and 470nm was measu ed. Chl a, b
and ca o enoids we e calcula ed using he ollowing equa-
ions (Lich en hale and Buschmann 2001):
An hocyanin concen a ion was measu ed dilu ing he
c ude ex ac wi h HCl un il he inal concen a ion eached
1% ( / ). Samples we e kep a 4 ºC in da kness. A e 24h,
Chl a(
𝜇
g∕ml)=16.72 Abs665.2 −9.16 Abs652.4
Chl b(
𝜇
g∕ml)=34.09 Abs652.4 −15.28 Abs665.2
Ca
(𝜇g∕ml)=
(
1000 Abs470 −1.63 Chl a−104.96 Chl b
)
∕
221
Plan a (2025) 261:9999 Page 4 o 17
abso bance was measu ed a 530nm and 653nm. An hocya-
nin concen a ion was de e mined acco ding o Mancinelli
e al. (1974). These and all o he UV/ isible spec opho-
ome ic assays desc ibed below we e ca ied ou using a
UV-3100PC spec opho ome e (VWR).
Polyphenols
Many plan species syn hesize and accumula e phenolic
compounds as a de ense mechanism agains en i onmen-
al s esso s (Yang e al. 2018). Phenolic compounds we e
assayed using he Folin-Ciocal eu eagen ollowing Single-
on and Rossi (1965). To assess polyphenol concen a ions,
0.5ml o Folin–Ciocal eau and 4.5ml o dis illed wa e we e
added o 1ml o c ude ex ac (p e iously dilu ed in dis-
illed wa e in a 7:3 a io). Samples we e pu in da kness a
oom empe a u e. A e 8min, 4ml o 7.5% (w/ ) sodium
ca bona e (Na2CO3) was added o each sample. A e 1-h
incuba ion unde he same condi ions abso bance a 765nm
was measu ed. Polyphenol concen a ions we e exp essed
in mg o gallic acid (GA) g DW−1 (Slama e al. 2017).
GA was used as a s anda d compound. GA s ock solu ion
(20mg/100ml me hanol) was p epa ed and a ious dilu ions
we e ob ained o he s anda d calib a ion cu e.
To al an ioxidan capaci y
Reduc ion o Mo (VI) o Mo (V) a acid pH p oduces a
u quoise compound ha can be measu ed o assess o al
an ioxidan capaci y. To eco d o al an ioxidan capaci y
1ml o eagen (0.6M sul u ic acid, 28mM sodium phos-
pha e, 4mM ammonium molybda e) was added o 0.1ml o
c ude ex ac (p e iously dilu ed in dis illed wa e in a 7:3
a io). Samples we e kep a 95 ºC o 90min. A e cool-
ing o 20min a oom empe a u e, abso bance a 695nm
was measu ed agains a blank. To al an ioxidan capaci y
was exp essed in mg GA/g DW−1 (P ie o e al. 1999; Slama
e al. 2017).
Malondialdehyde de e mina ion
MDA, a bioma ke o lipid pe oxida ion, indica es oxida i e
damage esul ing om abio ic s esses (Kim e al. 2017).
To assess MDA concen a ion as a lipid pe oxida ion index,
we ollowed he p o ocol desc ibed by Hodges e al. (1999)
wi h some modi ica ions (Taula uo i e al. 2001). A o al
o 2.5ml o c ude ex ac was di ided in o wo es ubes,
each con aining 1.25ml. We added 1.5ml o 20% (w/ )
ichlo oace ic acid (TCA) o he i s ube, while he sec-
ond ube ecei ed 1.5ml o a solu ion con aining 0.5%
(w/ ) hioba bi u ic acid (TBA) in 20% (w/ ) TCA. Then,
0.25ml o pu e me hanol was added o each ube. A e a
15-min incuba ion a 95°C, samples we e placed on ice o
e mina e he eac ion. A e cen i uging samples a 9981g
o 10min, he supe na an 's abso bance was measu ed a
440nm, 532nm, and 600nm. MDA concen a ion was cal-
cula ed using he ollowing equa ions:
Apical lea g ow h
Apical lea g ow h se es as an e ec i e measu e o quan-
i ying s ess esponses in Spa ina axa in ield condi ions
(Cas illo e al. 2014). Apical lea g ow h was measu ed
ma king 3–5 lea es pe clump wi h pe manen sealan a
hei base, and measu ing he dis ance om he sealan o
he lea base 2days la e o he en ma ked clumps pe axon
(n = 10 samples pe axon).
Maximal a e o pho osyn he ic oxygen e olu ion
Maximal a e o pho osyn he ic oxygen e olu ion (Vmax) has
been p e iously eco ded o quan i y olia he mal s ess
in halophy es (Figue oa-Luque e al. 2024). We measu ed
Vmax o ob ain in o ma ion on he s a e o he pho osyn-
he ic appa a us unde o ced condi ions o CO2 sa u a ion
and high adia ion le el. Lea es we e sampled om he en
ma ked clumps pe axon in he e ening when he ide was
aising and jus be o e he s udy plan s we e being inun-
da ed (n = 10 samples pe axon). Then lea es we e s o ed
in a wa e apo -sa u a ed a mosphe e a 25°C a da k-
ness and 4 ºC un il measu emen s we e ca ied ou ea ly
in he mo ning. Vmax measu emen s we e ca ied ou using
an oxygen elec ode ype Cla k (Hansa ech LD2, Pen ney,
UK) a 25°C and pho osyn he ic pho on lux densi y (PPFD)
1400µmol m– 2 s−1 in a CO2-sa u a ed a mosphe e ob ained
wi h a 1M ca bona e/bica bona e bu e (pH = 9). Vmax
was eco ded as a elease o O2 pe uni o ime and g FW
(µmolO2 s−1 gFW−1) (n = 10 measu emen s pe axon) (Fa -
quha e al. 2001; Popo a e al. 2019).
Chlo ophyll luo escence
Chlo ophyll a luo escence is a use ul ool o assess he
e ec s o he mal s ess on he pho osyn he ic appa-
a us (Kune 2024). Ligh and da k-adap ed Chl luo-
escence we e measu ed in one lea pe each o he 10
ma ked clumps pe axon (n = 10 samples pe axon) a
sun ise (PPFD ca. 200µmol m−2 s−1) and a noon (PPFD
A
=Abs 532 −Abs 600 −
(
Abs 532
OnlyTCA
−Abs 600
OnlyTCA)
B=(Abs 440 −Abs 600)∗0.0571
MDA(nmol
mL )
=
(A−B
157000 )
∗10
6
Plan a (2025) 261:99 Page 5 o 17 99
o 1100–2100µmol m−2 s−1) wi h a po able modula ed
luo ime e (FMS-2, Hansa ech Ins umen s) using lea
clips o da k adap a ion o 30min. Noon measu e-
men s in o m abou he le els o o al (dynamic and pe -
manen ) pho oinhibi ion, while sun ise measu emen s
eco d he pe manen pho oinhibi ion le els ha has no
been able o eco e du ing he nigh (Fe nández-Baco
e al. 1998). Chl luo escence pa ame e s we e measu ed
acco ding o Maxwell and Johnson (2000). Ini ial luo es-
cence (F0) in he da k-adap ed s a e was measu ed using a
PPFD < 0.05µmol m–2 s−1 o 1.8µs, oo small o induce
signi ican physiological changes in he plan . Maximal
luo escence (Fm) was eco ded a e a sa u a ing ligh
pulse o 15.000µmol pho ons m−2 s−1. Va iable luo es-
cence (F = Fm − F0) and maximum quan um e iciency o
pho osys em II (PSII) pho ochemis y (F /Fm) we e cal-
cula ed o quan i y pho oinhibi ion. Using he same lea
sec ion, ligh -adap ed pa ame e s we e measu ed be o e
assessing da k-adap ed Chl luo escence in lea es accli-
ma ed o ull sola adia ion. S eady-s a e luo escence
yield (Fs) was eco ded a e adap ing plan s o ambien
ligh condi ions (wi h ull sunligh o 1150µmol pho ons
m−2 s−1). A sa u a ing ac inic ligh pulse o 15,000µmol
pho ons m−2 s−1 o 0.7s was hen used o p oduce he
maximum luo escence yield (Fm´) by empo a ily inhibi -
ing PSII pho ochemis y. E ec i e quan um e iciency o
PSII (ΦPSII = (Fm´—Fs)/Fm´) was calcula ed. Non-pho o-
chemical quenching (NPQ) was calcula ed om pa am-
e e s ob ained in bo h da k and ligh -adap ed s a es.
Da a analyses
Inhe i ance mechanisms
To in es iga e he inhe i ance mechanisms unde lying sal
s ess esponses, he abo e-desc ibed plan ai s we e ana-
lyzed in he ecip ocal hyb ids o S. ma i ima and S. densi-
lo a. The ollowing inhe i ance mechanisms we e iden i ied,
as desc ibed by Fa e and Ka enbe g (2011). (1) Dominan
inhe i ance, when a hyb id exhibi ed a ai simila o one o
i s pa en s. This was deno ed as “D-Sm” o S. ma i ima o
“D-Sd” o S. densi lo a. (2) Pa en al codominance when a
hyb id's ai was no di e en om he wo pa en s, ep e-
sen ed as “D-Sm,Sd”. (3) Pa en al addi i i y, when a hyb id
ai ell wi hin he ange o i s pa en s bu was signi ican ly
di e en om bo h. (4) T ansg essi e seg ega ion, when
hyb ids exhibi ed ai s ha exceeded, by a leas 5%, he
pheno ypic ange o bo h pa en s. These inhe i ance mech-
anisms we e quan i ied o each plan ai in bo h hyb id
popula ions and he di e ences be ween species we e based
on he signi ican di e ences exe ed o each a iable (see
nex sec ion).
S a is ical analyses
All s a is ical analyses we e ca ied ou using R so wa e
(R-co e eam 2024). We applied a signi icance le el (α) o
0.05 o e e y analysis. De ia ions o all da a we e calcu-
la ed as he s anda d e o o he mean (SE). Plan ai s
we e classi ied in o wo unc ional g oups: (1) biochemical
s ess esponses (an hocyanins, Chl a, Chl b, ca o enoids,
MDA, polyphenols and an ioxidan capaci y), and (2) eco-
physiological and g ow h esponses (F0, Fm, F , F /Fm, Fs,
Fm´, ΦPSII and NPQ a sun ise and noon, Vmax and apical lea
g ow h). To p o ec analyses om ype I e o , he means o
he dependen a iables o each ai g oup we e compa ed
using mul i a ia e analysis o a iance (MANOVA) and
Wilks’ Lambda o e alua e he signi icance o he ac o s
axon (S. ma i ima, S. densi lo a, S. ma i ima × densi lo a
and S. densi lo a × S. ma i ima) and season (win e and sum-
me ) (Scheine 2001). Redundan , highly co ela ed a i-
ables ( > 0.90) we e iden i ied p io o MANOVA analysis
and we e omi ed om he s a is ical models (Fm a sun ise
and noon). Once mul i a ia e signi icance was con i med
ia MANOVA, he main uni a ia e di e ences o plan
ai s we e e alua ed o each plan ai wi h Gene al Lin-
eal Models (GLMs) and Bon e oni-Dunn's es as a pos
hoc analysis. En i onmen al a iables we e also analyzed
using GLMs wi h axa and season as ixed ac o s and Bon-
e oni-Dunn's es as a pos hoc analysis. The adequacy o
model assump ions o he GLMs, including checking o
uni o mi y and esidual diagnos ics, was assessed using he
DHARMa package (Ha ig 2022). En i onmen al a iables
we e also compa ed be ween seasons and axa using GLMs
and Bon e oni-Dunn's es as a pos hoc analysis.
We pe o med Canonical Co espondence Analyses
(CCA), using he egan package (Oksanen e al. 2024), o
iden i y he en i onmen al a iables ha mos g ea ly in lu-
enced plan ai s in he s udied Spa ina axa. CCA was
conduc ed using a ull model o es he signi icance o he
ela ionships be ween he en i onmen al a iables measu ed
in win e and summe and he plan ai s ma ix o each
axon. Mon e-Ca lo pe mu a ion es s (999 pe mu a ions)
we e pe o med o assessing he signi icance o he canon-
ical co ela ion coe icien s. A e age daily maximum and
minimum ai empe a u es we e emo ed du ing he analysis
due o hei mul icollinea i y wi h a e age daily mean ai
empe a u e.
Resul s
En i onmen al ma ix
The s udy a ea p esen ed ma ked seasonal ai em-
pe a u es and ain all was concen a ed mainly om

Plan a (2025) 261:9999 Page 6 o 17
Sep embe o Feb ua y du ing he sampling yea (Fig.
S2). Mean minimum daily ai empe a u e was 21 ºC
lowe du ing he win e cold snap han in he summe
hea wa e. This seasonal di e ence was 10 ºC o a e -
age daily empe a u e and 19 ºC o maximum daily
empe a u e. Mean sedimen Eh was always highe han
95mV o e e y axon in bo h seasons, being highe in
win e han in summe . Sedimen WC did no p esen
seasonal di e ences, and a ied be ween 45 ± 1% o S.
densi lo a × ma i ima in win e and 72 ± 2% o S. ma -
i ima × densi lo a in summe . Sedimen EC was highe
in summe han in win e , and a ied be ween 8.7 ± 0.5
mS cm−1 o S. densi lo a in win e and 16.7 ± 0.5 mS
cm−1 o S. densi lo a × ma i ima in summe . Mean sedi-
men pH was highe in win e han in summe and anged
be ween 6.7 and 7.5 (Tables1 and 2).
Plan esponses
Spa ina ma i ima, S. densi lo a and bo h ecip ocal
hyb ids exhibi ed seasonal a ia ions in di e en bio-
chemical, ecophysiological and g ow h ai s (Tables3
and 4).
Pigmen s
The ou Spa ina axa exhibi ed highe an hocyanin concen-
a ion in win e han in summe , wi h S. densi lo a show-
ing a lowe inc ease han he hyb ids (Fig.1A). Spa ina
densi lo a and S. densi lo a × ma i ima p esen ed lowe
an hocyanin concen a ions han S. ma i ima and S. ma -
i ima × densi lo a (TableS1). Spa ina ma i ima p esen ed
highe Chl a concen a ion and lowe ca o enoids concen-
a ion in summe han in win e (Fig.1B, D). Spa ina
ma i ima accumula ed mo e ca o enoids in win e han S.
Table 1 F-s a is ic and P- alues
o Gene al Linea Models
wi h axon (T), season (S)
(win e and summe ) and hei
in e ac ion (T x S) as ixed
ac o s, o en i onmen al
a iables measu ed in
indi iduals o Spa ina
ma i ima, Spa ina densi lo a
and hei ecip ocal hyb ids
in he Guadiana Ma shes
(Sou hwes Ibe ian Peninsula)
Values o F-s a is ics wi h deg ees o eedom (n) as subsc ip s a e displayed. Signi ican di e ences a e
highligh ed in bold
En i onmen al a iables Taxon (T) Season (S) T × S
F3,72 PF1,18–72 PF3,72 P
Minimum daily ai empe a u e (ºC) – – 294.63 < 0.0001 – –
A e age daily ai empe a u e (ºC) – – 873.15 < 0.0001 – –
Maximum daily ai empe a u e (ºC) – – 536.83 < 0.0001 – –
Sedimen edox po en ial (mV) 3.888 0.012 4.657 0.034 0.267 0.849
Sedimen wa e con en (%) 37.139 < 0.0001 1.105 0.297 4.534 0.006
Sedimen elec ical conduc i i y (mS cm−1)16.204 < 0.0001 11.921 0.001 0.017 0.997
Sedimen pH 52.956 < 0.0001 50.579 < 0.0001 2.403 0.075
Table 2 Me eo ological and sedimen a y en i onmen al ac o s o na i e Spa ina ma i ima, in oduced S. densi lo a and hei ecip ocal
hyb ids in he Guadiana Ma shes (Sou hwes Ibe ian Peninsula)
Values a e mean ± SE (n = 10). Di e en le e s indica e signi ican seasonal di e ences o a gi en axon (Gene al Lineal Models and Bon e -
oni-Dunn's es as a pos hoc analysis; see Table1)
Win e Summe
S. ma i ima S. ma -
i ima × den-
si lo a
S. densi lo a S. densi-
lo a × ma -
i ima
S. ma i ima S. ma -
i ima × den-
si lo a
S. densi lo a S. densi-
lo a × ma -
i ima
Minimum daily ai empe a u e
(ºC)
4.0 ± 0.6a25.2 ± 0.4b
A e age daily ai empe a u e
(ºC)
8.8 ± 0.4a18.7 ± 0.6b
Maximum daily ai empe a u e
(ºC)
13.3 ± 0.6a31.7 ± 0.5b
Sedimen edox po en ial (mV) 167 ± 22 175 ± 24 189 ± 18 125 ± 11 124 ± 21 168 ± 28 149 ± 18 96 ± 15
Sedimen wa e con en (%) 70 ± 2 70 ± 2 71 ± 2 45 ± 1 67 ± 2 72 ± 2 68 ± 3 56 ± 2
Sedimen pH 7.5 ± 0.0 7.4 ± 0.0 7.5 ± 0.0 6.9 ± 0.0 7.1 ± 0.1 7.3 ± 0.1 7.2 ± 0.1 6.7 ± 0.0
Sedimen elec ical conduc i i y
(mS cm−1)
9.1 ± 0.7 9.0 ± 0.9 8.7 ± 0.5 14.5 ± 1.0 11.3 ± 1.1 11.4 ± 1.3 11.2 ± 1.2 16.7 ± 0.5
Plan a (2025) 261:99 Page 7 o 17 99
densi lo a and S. densi lo a × ma i ima (TableS1). Chl b
concen a ion did no change signi ican ly be ween seasons
o any axa (Fig.1C).
MDA, an ioxidan capaci y andpolyphenols
The hyb id S. ma i ima × densi lo a was he only axon ha
p esen ed a highe (+ 68%) MDA concen a ion in sum-
me han in win e (Fig.1E). Spa ina ma i ima × densi-
lo a accumula ed less MDA han S. ma i ima in win e ,
wi h S. densi lo a and S. densi lo a × ma i ima p esen ing
in e media e alues (TableS1). Bo h hyb ids p oduced a
highe (+ 35%) amoun o polyphenols (Fig.1F) and had
g ea e (+ 47%) o al an ioxidan capaci y in win e com-
pa ed o summe . Spa ina ma i ima also exhibi ed highe
(+ 24%) o al an ioxidan capaci y in win e han in summe
(Fig.1G). In win e , S. ma i ima accumula ed mo e poly-
phenols han S. densi lo a × ma i ima and S. densi lo a. To al
an ioxidan capaci y was lowe o S. densi lo a in win e
and o S. ma i ima × densi lo a in summe han o he o he
h ee axa (TableS1).
G ow h andpho osyn hesis
Apical lea g ow h was 38% highe in summe han
in win e . This seasonal change was eco ded o
e e y axon, being signi ican o S. densi lo a and S.
Table 3 Wilks’ lambda, F-s a is ic, deg ees o eedom and P- alues
om MANOVAs o he wo ai esponse g oups o he ac o s sea-
son (win e and summe ), axon (S. ma i ima, S. densi lo a, S. ma -
i ima × densi lo a and S. densi lo a × ma i ima) and hei in e ac ion
The highe Wilks’ lambda is, he s onge he e idence ha he inde-
penden a iables ( ac o s) ha e a s a is ically signi ican e ec on he
dependen a iable
Fac o s Wilks’
Lambda
F d P
Biochemis y Season 0.165 18.832 7 < 0.001
Taxon 0.023 0.984 21 < 0.001
Season*Taxon 0.057 6.120 21 < 0.001
Ecophysiol-
ogy and
g ow h
Season 0.042 81.726 16 < 0.001
Taxon 0.094 4.308 48 < 0.001
Season*Taxon 0.233 2.240 48 < 0.001
Table 4 F-s a is ic and P- alues
o Gene al Linea Models
wi h axon (T), season (S)
(win e and summe ) and
hei in e ac ion (T x S) as
ixed ac o s, o biochemical
and ecophysiological and
g ow h- ela ed ai s measu ed
in indi iduals o Spa ina
ma i ima, Spa ina densi lo a
and hei ecip ocal hyb ids
in he Guadiana Ma shes
(Sou hwes Ibe ian Peninsula)
Signi ican di e ences a e highligh ed in bold
Plan ai s Taxon (T) Season (S) T × S
F3,32 PF1,32 PF3,32 P
Biochemis y An hocyanins (µg g DW−1)9.28 < 0.001 49.33 < 0.001 2.05 0.126
Chl a (µg g DW−1)12.45 < 0.001 12.00 < 0.01 2.17 0.111
Chl b (µg g DW−1)4.82 < 0.001 2.29 0.140 0.68 0.572
Ca o enoids (µg g DW−1)8.17 < 0.001 17.69 < 0.001 10.90 < 0.001
MDA (nmol g DW−1) 2.263 0.100 4.58 < 0.05 6.13 < 0.01
Polyphenols (mg g DW−1)66.12 < 0.001 15.81 < 0.001 6.08 < 0.01
An ioxidan capaci y (mg g DW−1)17.75 < 0.001 71.53 < 0.001 20.62 < 0.001
F3,72 PF1,72 PF3,72 P
Ecophysiol-
ogy and
g ow h
Apical lea g ow h (cm) 21.37 < 0.001 35.68 < 0.001 0.54 0.653
Vmax (μmol O2 g−1 FW−1)6.20 < 0.001 26.16 < 0.001 0.61 0.608
F0 sun ise 2.77 < 0.05 1.49 0.226 2.00 0.121
F sun ise 6.57 < 0.001 22.83 < 0.001 5.78 < 0.01
F /Fm sun ise 6.22 < 0.001 13.91 < 0.001 1.29 0.284
Fs sun ise 2.90 < 0.05 9.28 < 0.01 4.31 < 0.01
Fm’ sun ise 3.09 < 0.05 103.30 < 0.001 6.89 < 0.001
ΦPSII sun ise 6.36 < 0.01 726.75 < 0.001 3.39 < 0.05
NPQ sun ise 1.44 0.238 25.19 < 0.001 2.50 0.066
F0 noon 0.85 0.470 7.53 < 0.01 0.89 0.451
F noon 4.32 < 0.01 21.16 < 0.001 4.64 < 0.01
F /Fm noon 2.63 0.056 4.12 < 0.05 2.38 0.077
Fs noon 2.21 0.094 1.63 0.205 5.22 < 0.01
Fm’ noon 2.07 0.112 0.55 0.459 4.80 < 0.01
ΦPSII noon 1.01 0.392 15.62 < 0.001 1.47 0.229
NPQ noon 1.13 0.341 3.32 0.0725 3.80 < 0.05
Plan a (2025) 261:9999 Page 8 o 17
ma i ima × densi lo a (Fig.2A). Vmax ended o be highe
in win e han in summe o e e y axon, al hough his
seasonal a ia ion was only signi ican o S. densi-
lo a × ma i ima (Fig.2B). Spa ina ma i ima p esen ed
highe Vmax, bu g ew less han he o he h ee axa
(TableS1).
Chlo ophyll luo escence
Spa ina ma i ima × densi lo a was he only axon showing
highe (+ 9%) F /Fm a sun ise in summe han in win-
e (Fig.2E), h ough a ma ked inc ease (+ 46%) in F
(Fig.2D). ΦPSII a sun ise was ma kedly highe (+ 67%)
Fig. 1 Seasonal di e ences in biochemical ai s o Spa ina ma -
i ima (Sm, black column), Spa ina densi lo a (Sd, whi e column)
and hei wo ecip ocal hyb ids, S. ma i ima × densi lo a (Smxd, da k
g ey column) and S. densi lo a × ma i ima (Sdxm, ligh g ey column).
Values a e means ± SE (n = 5). Nega i e alues indica e highe mag-
ni udes in win e and posi i e alues indica e highe magni udes in
summe . As e isks deno e seasonal signi icance a P < 0.05 (GLM)
Plan a (2025) 261:99 Page 9 o 17 99
in summe han in win e o all axa (Fig.2H) due o
highe Fm’ (Fig.2G), excep o S. densi lo a ha showed
highe (+ 107%) Fs in win e han summe (Fig.2F). Bo h
hyb ids p esen ed highe ΦPSII a sun ise han S. ma i ima
in summe . Addi ionally, he wo hyb ids showed highe
NPQ a sun ise in win e han in summe , wi h S. ma i -
ima × densi lo a exhibi ing he g ea es (+ 355%) seasonal
inc ease (Fig.2I) (TableS1).
When Chl luo escence measu emen s we e eco ded
a noon, S. ma i ima × densi lo a exhibi ed he majo i y o
he seasonal a ia ions. This hyb id showed highe (+ 9%)
F /Fm [associa ed wi h inc eased (+ 46%) F ], and lowe
(−229%) ΦPSII (wi h inc eased Fs and Fm’ alues) in
summe han in win e (Fig.3B–F). S. densi lo a was he
only axon exhibi ing highe (+ 59%) NPQ in summe han
Fig. 2 Seasonal di e ences in apical lea g ow h (A) and Vmax (B),
and F0 (C), F (D), F /Fm (E), Fs (F), Fm’ (G), ΦPSII (H) and NPQ
(I) a sun ise o Spa ina ma i ima (Sm, black column), Spa ina
densi lo a (Sd, whi e column) and hei wo ecip ocal hyb ids, S.
ma i ima × densi lo a (Smxd, da k g ey) and S. densi lo a × ma i ima
(Sdxm, ligh g ey). Values a e means ± SE (n = 10). Nega i e alues
indica e highe magni udes in win e , and posi i e alues indica e
highe magni udes in summe . As e isks deno e seasonal signi icance
a P < 0.05 (GLM)
Plan a (2025) 261:9999 Page 16 o 17
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Publishe 's No e Sp inge Na u e emains neu al wi h ega d o
ju isdic ional claims in published maps and ins i u ional a ilia ions.