En i onmen al and Expe imen al Bo any 194 (2022) 104738
A ailable online 1 Decembe 2021
0098-8472/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license
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Hea y me al ole ance s a egies in me allicolous and non-me allicolous
popula ions o mosses: Insigh s o γ+β- ocophe ol egula o y ole
An onio De Agos ini
a
, Annalena Cogoni
a
, Pie luigi Co is
a
, And ea Vacca
b
,
Jos´
e Ma ía Bece il
c
, An onio He n´
andez
c
, Raquel Es eban
c
,
*
a
Depa men o Li e and En i onmen al Sciences, Uni e si y o Caglia i, Via San ’Ignazio 13, 09123 Caglia i (CA), I aly
b
Depa men o Chemical and Geological Sciences, Uni e si y o Caglia i, Ci adella Uni e si a ia (Blocco D), S.S. 554, 09042 Monse a o (CA), I aly
c
Depa men o Plan Biology and Ecology. Uni e si y o he Basque Coun y (UPV/EHU), 48080 Bilbao, Spain
ARTICLE INFO
Keywo ds:
B yological a ibu es
Mining soils
B yophy es
Pigmen s
Tocophe ols
Tole ance
ABSTRACT
As b yophy es p esen he capaci y o colonize a wide ange o habi a s, including ex eme en i onmen s (e.g.,
mine a eas), we i s checked he global esponse o b yophy es o hea y me als (HM) by a sys ema ic e iew.
This analysis ound i) he lack o s udies in a pollu ed en i onmen and ii) esul ed in a global end o pho o-
chemical e iciency dec ease when b yophy es aced hea y me als. Secondly, we cha ac e ized he pho o-
p o ec i e esponses o ou me allicolous popula ions o mosses (Lewinskya upes is, Poly ichum commune,
P ychos omum compac um and Rhynchos egium con e um) na u ally g owing in an abandoned mine a ea by an
expe imen al ield app oach. As a esul , we ound ma kedly species-speci ic ole ance pa e ns: i) less ole an
species, p esen ing high pollu an s con en accompanied by ma ked dissipa i e and an ioxida i e s a egies. This
s a egy was depic ed by he species R. con e um ha esul o be a good sen inel species due o he high
pollu an con en and high physiological sensi i i y.; ii) in e media e ole an species p esen ing a medium
con en o pollu an s and less ma ked pho op o ec i e mechanisms, and iii) mos ole an species minimizing
pollu an s con en and consequen ly p esen ing no changes in hei physiological pe o mance. This e idence
suppo ha species’ b yological a ibu es ha e a key ole in de e mining species’ ole ance owa ds en i on-
men al hea y me als, and should be aken in o conside a ion in u u e s udies. The indings o his s udy also
poin ed ou ha he highe le els o γ +β- ocophe ol in me allicolous popula ions could play a egula o y ole in
me al s ess ole ance in mosses and i could be a sui able unc ional esponsi e ai o en i onmen al esponse
p edic ion o hea y me als in pollu ed en i onmen s.
1. In oduc ion
Abandoned mining a eas, as impo e ished heaps and ailing dumps,
ep esen a se ious en i onmen al p oblem due o he high p esence o
ino ganic pollu an s, such as hea y me als (HM) and me alloids, e en
when ex ac ion has long ceased o be ac i e (Vacca and Vacca, 2001;
Bacche a e al., 2015). These compounds can each high concen a ion
le els and bioa ailabili y in soils and e en in soil o ganisms, as hey do
no unde go ecosys em deg ada ion (Ba u ia e al., 2011). These
an h opogenic soil ma ices esul o be sca cely colonized by plan s due
o he challenge o li e unde he abio ic s esses ha hese pollu ed
en i onmen s p esen such as poo ly consolida ed g ow h subs a e,
sho age o o ganic ma e and nu ien s, phy o oxici y o soil pollu an s
and high sun i adia ion due o he absence o a ege al canopy (Ba u ia
e al., 2011; Jim´
enez e al., 2011; Bacche a e al., 2018; De Agos ini
e al., 2020a). Mo e pa icula ly, non-adap ed plan s can su e a s ong
de imen al HM- ela ed phy o oxic e ec on physiology and li e cycle,
nega i ely in e e ing wi h essen ial p ocesses as pho osyn hesis,
Abb e ia ions: A, an he axan hin; β-ca o , β-ca o ene; Chl, chlo ophyll; CB, con amina ed b yophy es; DW, d y weigh ; E1, majo biome; E2, he eas e n limi o
dis ibu ion; FW, esh weigh ; F, mois u e; HM, hea y me als; HM , ole ance o HM; L, ligh ; Len, game opho es size; LF1, li e- o m; Lu , lu ein; MP, me allicolous
popula ion; NCB, non-con amina ed b yophy es; NMP, non-me allicolous popula ion; N, ni ogen; Neox, neoxan hin; NPQ, non-pho ochemical quenching; PCA,
p incipal componen analysis; Pe , ype o pe enna ion; R, eac ion o en i onmen al acidi y; ROS, eac i e oxygen species; Sex, dis ibu ion o game angia in
game opho e; TAC, o al an ioxidan capaci y; V, iolaxan hin; Z, zeaxan hin.
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (R. Es eban).
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En i onmen al and Expe imen al Bo any 194 (2022) 104738
2
espi a ion, nu ien up ake, gene exp ession and memb ane in eg i y
(Baek e al., 2012). Mo eo e , some me als (Fe, Cu, Mo, e c.) can in e ac
wi h cellula oxygen o o m eac i e oxygen species (ROS), change cell
edox s a us, and e en ually eplace me allic co ac o s in unc ioning
enzymes inhibi ing hei unc ionali y (Baek e al., 2012). Consequen ly,
ascula plan s own se e al mechanisms o ole a e HM s ess and su -
i e in such pollu ed en i onmen s and, as a esul , hese me allicolous
popula ions bene i om he educed compe ence o o he plan s in
hese hos ile en i onmen s (She e son e al., 2008; Faucon e al., 2011;
De Agos ini e al., 2020a). The mechanisms o ole ance o ascula
plan s a e widely s udied (Baek e al., 2012; Chand a and Kang, 2015;
Singh e al., 2015). On he con a y, he mechanisms in ol ed in he
esponse o HM in non- ascula plan s, as b yophy es, a e no so well
cha ac e ized.
B yophy es a e emb yophy e plan s ha di e om acheophy es in
se e al ana omical and physiological ai s: hey lack a oo sys em
( hizoids mainly unc ion consis in ancho ing indi iduals o he sub-
s a e), as well as an e icien conduc ion sys em (hyd oids and lep oids
desc ibed in Poly ichum spp., Spagnum spp., e c. no as e icien as he
conduc ion sys ems in ascula lo a;Lig one e al., 2000). Those ea-
u es imply ha b yophy es nu ien s’ acquisi ion in o game ophy es
elies almos exclusi ely on d y and we deposi ion and secondly, on
abso p ion om he subs a um ia capilla i y (Chak abo y and Pa -
a ka , 2006). To op imize his o m o nu ien acquisi ion, b yophy es
a e cha ac e ized by a g ea su ace- o- olume a io. Phylloids indeed
a e gene ally cons i u ed by a single laye o cells and hey do no p e-
sen any kind o physical, chemical, o cellula ba ie p e en ing wa e
en ance o loss such as epide mis, cu icles, o s oma a. As a esul ,
b yophy es a e cha ac e ized by a g ea pe meabili y o wa e and ele-
men s dissol ed in i (Chak abo y and Pa a ka , 2006; De Agos ini
e al., 2020b). Despi e he disad an ages linked o poo con ol o e
nu ien up ake and wa e loss, b yophy es a e p esen in e e y con i-
nen and a e well adap ed o li e in se e al ecological con ex s,
including he ex eme ones, in which hey gene ally ac as ea ly colo-
nize s (Holyoak, 2009). Fo example, b yophy es a e o en desc ibed as
colonizing HM pollu ed si es, such as abandoned mining a eas (Holyoak,
2009; Holyoak and Lockha , 2011; Ka akaya e al., 2015; Angelo ska
e al., 2016) and some moss species, such as Scopelophila ca a ac ae
(Mi .) B o h., a e conside ed o be me al- ole an species (Shaw, 1987;
Konno e al., 2010; Yagu a e al., 2019).
Due o he physiological and mo phological ea u es o b yophy es
and hei high exposu e o d y and we deposi ion, his g oup is la gely
used in en i onmen al pollu an s’ biomoni o ing (A es e al., 2014;
Chak abo y and Pa a ka , 2006; Co is e al., 2016; De Agos ini e al.,
2020a; Esposi o e al., 2018; Ma esca e al., 2020, 2018). Ne e heless,
besides a ecen e iew a icle (S anko i´
c e al., 2018), s udies o en i-
onmen al HM pollu ion’s e ec s on ecology, mo phology, and physi-
ology on b yophy es a e no so equen . Mos o he scien i ic li e a u e
on he opic epo s ha : i) b yophy es can accumula e pollu an s on
hei su ace, adso b hem on cells’ walls and, ac i ely o passi ely,
anspo hem in o cells (Chak abo y and Pa a ka , 2006; Basile e al.,
2012); ii) physiochemical ea u es o pollu an s (pa icula e ma e size,
chemical na u e o pollu an s, as well as hei solubili y and cha ge)
s ongly in luence hei accumula ion in b yophy es (Chak abo y and
Pa a ka , 2006; Basile e al., 2012; Capozzi e al., 2020); iii) se e al e -
ec s, o en dose-dependen , on b yophy es’ physiology ha e been
ela ed o en i onmen al pollu ion, he mos ele an nega i ely in lu-
encing pho osyn he ic pa ame e s, pigmen con en , cellula ul a-
s uc u al o ganiza ion, p o ein con o ma ion and biosyn hesis a es,
and memb ane in eg i y (Shakya e al., 2008; Basile e al., 2012; Co is
e al., 2016; S anko i´
c e al., 2018; Chen e al., 2019), and i ) in a-
speci ic di e ences and e en sexual dimo phism we e obse ed o in-
luence ole ance o en i onmen al HM in b yophy es (Boque e e al.,
2021).
As ega ds he b yophy es adap a ions o HM pollu ed en i onmen s,
hey ha e been desc ibed o consis in: i) accumula ion o oxic elemen s
in cell walls (K zesłowska e al., 2009; Konno e al., 2010; Ka akaya
e al., 2015) and acuoles (Basile e al., 1994; Ca ginale e al., 2004); ii)
exc e ion o HM sal s om game ophy e (Rao, 1982; Tyle , 1990); iii)
spo es p o ec ion om pollu an s (Basile e al., 2001); and i ) p oduc-
ion o an ioxidan s (Bellini e al., 2020; Ma esca e al., 2020; Vuo o
e al., 1991, 2002; Ielpo e al., 1998)and hea shock p o eins and phy-
ochela ins (Basile e al., 2004; Ca ginale e al., 2004). Ne e heless, he
ole o pho op o ec i e mechanisms o b yophy es o HM is e y poo ly
s udied, especially in me allicolous popula ions o mining a eas.
We hypo hesized ha pho op o ec ion and an ioxidan mechanisms
(specially pigmen s and ocophe ols) play a key ole in b yophy es’
adap a ion o HM s ess, allowing his componen o he lo a o cope
wi h HM soil pollu ion. B yophy es as pionee plan s species could play
an impo an ecological se ice in he es o a ion o soil e ili y,
inc easing soil o ganic ma e and a ou ing ascula plan s and mi-
c obes’ coloniza ion and biodi e si y in such hos ile en i onmen s as
me alli e ous si es a e (Sza ek-Łukaszewska, 2009; Ba u ia e al., 2011;
Holyoak and Lockha , 2011; Callaghan, 2018). In his s udy, he e o e,
we i s checked he global esponse o b yophy es o HM by a da a
compila ion om li e a u e ( om 1999 o 2019), and secondly, we
compa ed he ele an ecological/physiological ai s ha e ol ed o
colonize mining en i onmen s in ou moss species (wi h con as ing
b yological a ibu es) g owing na u ally on me alli e ous and
non-me alli e ous a eas. We analysed he physiological esponse by he
ollowing ai s: physiological pe o mance (pho osyn he ic e iciency)
and biochemical pa ame e s (chlo ophylls, ca o enoids, ocophe ols,
o al an ioxidan capaci y, and an hocyanins con en , as well as olia
pH), oge he wi h he main b yophy e ecological a ibu es. We aimed
o answe he ollowing ques ions: i) Do ai s endency ob ained om
he analysis o da a om li e a u e mi o he ai endency ob ained in
he ield? ii) Is HM ole ance species- ela ed showing each
species-speci ic ole ance ai ? iii) Do pho op o ec i e compounds play
an impo an ole in b yophy es ole ance o HM oxici y? This s udy
will p o ide impo an clues ega ding ecophysiological ai s ac i a ed
in b yophy es me allicolous popula ions o mining a eas.
2. Ma e ials and me hods
i) We i s analysed li e a u e da a on he pho op o ec i e esponse o
mosses and ii) second, we pe o med an expe imen al ield app oach
compa ing me allicolous and non-me allicolous popula ions. See he
de ails below.
2.1. Analysis o da a compila ion om he li e a u e
2.1.1. Li e a u e sea ch and inclusion c i e ia
Da a we e collec ed exclusi ely om pee - e iewed jou nals pub-
lished in he wo decades om 1999 o 2019, using he esou ce “ISI
Web o Knowledge”. Que ies we e made using Boolean ope a o s (AND,
OR and NOT) wi h combina ions o he wo ds: “b yophy e”, “moss”,
“pigmen ”, “ca o enoid”, “ ocophe ol”, “chlo ophyll”, “an ioxidan ”,
“pho osyn hesis”, “hea y me al”, “soil pollu ion” and “s ess”. As a
esul o his sea ch c i e ion, a o al o 561 a icles we e ound. A e
his i s sea ch, a inal da abase was c ea ed o he analysis phase
applying he ollowing inclusion c i e ia: pape s dealing wi h s ess
ac o s di e ing om HM (e.g., pes icides, he bicides, ammonium, e c.)
we e no conside ed; a icles wi hou men ion o b yophy es o
conside ing o he axa, eg. lichens, Spanish moss (Tillandsia usneoides
L.), e c., we e excluded; a icles conside ing only ul as uc u al e ec s
o an ioxidan enzymes, e.g., supe oxide dismu ase, pe oxidase, e c.,
we e no aken in o accoun ; e iew- ype a icles and me hodological
a icles we e also excluded. In his way, a icles sui able o be used in he
analy ical phase esul ed o be in he numbe o 35 (lis ed in Table 1S),
including 38 di e en species o b yophy es (among hose 6 belonging
o he Ma chan iophy a di ision), di ided in o 19 amilies and 13 o de s
(Table 2S).
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
3
2.1.2. Da abase c ea ion and analy ical p ocedu e
Da a we e ex ac ed om ables and igu es o he 35 selec ed a icles
wi h he p og am ImageJ e sion 1.52a (Schneide e al., 2012). To
del e in o he di e ences in pho op o ec i e mechanisms due o HM,
wo ca ego ies we e c ea ed: i) con amina ed b yophy es (he eina e
CB), conside ed hose exposed o he mos in ense o p olonged HM
s ess, and ii) non-con amina ed b yophy es (he eina e NCB), consid-
e ed hose indica ed so in he o iginal s udies. When hese alues lacked
in he o iginal a icle, b yophy es g owing unde he mino g ade o HM
s ess we e conside ed as NCB. The ollowing pa ame e s we e included
in he da abase: he maximal pho ochemical e iciency o pho osys em II
(F
V
/F
M
), he non-pho ochemical quenching (NPQ), chlo ophyll a (Chl
a), chlo ophyll b (Chl b), o al chlo ophyll (Chl a+b), chlo ophyll a o b
a io (Chl a/b), o al ca o enoids, ca o enoids o Chl a+b a io (ca -
o enoids/Chl), an hocyanins and asco ba e. When any o hese da a
we e missing, and whene e possible, hey we e calcula ed om he
o iginal da a om he pape . In some cases, uni s we e con e ed. As his
con e sion was impossible in many cases, o s anda dize he di e si y o
da a uni s ex ac ed om he di e en a icles, we exp essed he inal
alues as a ia ions in pe cen age wi h espec o NCB alues, calcula ed
as (CB/NCB-1) x 100.
2.2. Expe imen al ield app oach
2.2.1. Field si es desc ip ion
We selec ed wo si es, an abandoned mining a ea and a con ol a ea,
bo h loca ed in he municipali y o Ca anza (Basque Coun y, Spain) a
empe a e egion in No he n Spain (A lan ic egion), in which he
annual ain all eaches 1400 mm and he annual mean empe a u e
anges be ween 11 and 15 ◦C (Galende e al., 2014). The me alli e ous
si e (43◦13′39.75 N, 3◦25′50.75′’ W), an abandoned lead (Pb) and zinc
(Zn) mine was chosen as i p esen s open pi s, was e ocks, and HM
pollu ed ailings dams (Ba u ia e al., 2011), and i is colonized by
se e al b yophy e species. Mean o al concen a ions o Zn, Pb and Cd in
he soil we e 30,850, 12,300 and 26 mg/kg espec i ely. The ex ac able
ac ion (CaCl
2
ex ac able) was 656, 21 and 2 mg/kg o Zn, Pb and Cd
espec i ely (Ba u ia e al., 2011). Mining ac i i y in he a ea ceased in
he 1970 s and nowadays he si e appea s na u ally colonized by a wide
ange o me allicolous popula ions o , among o he s, Noccaea cae -
ulescens J.P esl & C.P esl F.K.Mey., Ulex eu opaeus Sa i, Ag os is capil-
la is L. and Fes uca ub a L. (Ba u ia e al., 2011). The non-me alli e ous
si e (43◦13′57.79′’ N, 3◦26′24.49′’ W) was chosen in an a ea non
a ec ed by mine ac i i ies close o he me alli e ous si e (≈1 km om
he me alli e ous si e) and p esen ing simila edaphoclima ic condi ions,
excep o he p esence o me als in he soil.
2.2.2. Sample collec ion, iden i ica ion and s o age
The sampling o plan ma e ial was pe o med du ing May 24 h,
2019 (a non-s ess ul mon h) in he abandoned mining a ea and he
close con ol a ea. We andomly collec ed a midday se e al b yophy es
ca pe s (1 cm
2
) om h ee sepa a ed loca ions in he abandoned mining
a ea and he con ol a ea, paying a en ion o p ese ing he na u al
popula ions and pooling hem o ob ain a homogenous sample o each
g ow h condi ion o each species. The collec ed ma e ial was s o ed in
he da k in zip-locked polyp opylene bags wi h humidi y kep a high
le els and anspo ed o he labo a o y in a po able icebox ( empe a-
u e below 5 ◦C) (Fe n´
andez-Ma ín e al., 2018). Once in he labo a o y,
samples we e kep in da kness in a sa u a ed a mosphe e o 12 h o
p o ide compa able condi ions (Tausz e al., 2003). Heal hy g een
apices (1–3 cm long, depending on he species) we e cu and epea edly
cleaned om any deb is and washed wi h demine alized wa e un il he
comple e emo al o any es o soil and pa icula e ma e be o e
u he analysis. Iden i ica ion o he collec ed species was ca ied ou
on ai -d ied samples in he labo a o y, ollowing he mos ecen and
adequa e iden i ica ion dicho omous keys (Casas e al., 2006). The
species we e iden i ied as Lewinskya upes is (Schleich. ex Schw¨
ag .) F.
La a, Ga ille i & Go ine (O ho ichaceae), Poly ichum commune
Hedw. (Poly ichaceae), P ychos omum compac um Ho nsch. (B yaceae),
Rhynchos egium con e um (Dicks.) Schimp. (B achy heciaceae), as e-
po ed in Hodge s and Lockha (2020) and Hodge s e al. (2020). Fo
each sampled species, 3–5 eplica es o abou 20 mg esh weigh (FW)
o pho osyn he ic issue o each popula ion om he me alli e ous and
non-me alli e ous si es we e d ied a −80 ◦C o 48 h o ozen wi h
liquid ni ogen (depending on he analysis o be pe o med) and s o ed
un il u he use. As ai s in b yophy es will depend on he hyd a ion
s a us, we calcula ed he samples’ d y weigh (DW).
2.2.3. Species’ b yological a ibu es and au ecology
B yological a ibu es and au ecology o each moss species we e
desc ibed (Table 1) using he BRYOATT da abase ollowing Hill and
P es on (1998) and Hill e al. (2007) o compile o each species ai s
in o ma ion. These species we e classi ied acco ding o li e- o m (LF1),
ype o pe enna ion (Pe ), dis ibu ion o game angia in game opho e
(Sex), game opho es size (Len), majo biome (E1) and eas e n limi o
dis ibu ion (E2). The ollowing indices, each one ep esen ing di e en
ecological species’ equi emen s, we e also de e mined: ligh (L),
mois u e (F), eac ion o en i onmen al acidi y (R), ni ogen alues (N)
and ole ance o HM (HM ). The desc ip ion o he indices is lis ed
below. E1, anges om 1 (A c ic, mon ane biome) o 9 (Medi e anean,
A lan ic biome). E2, goes om 0 (hype oceanic) o 6 (ci cumpola ). LF1
is desc ibed by he ollowing ca ego ies: Ac, aqua ic colonial; A , aqua ic
ailing; Cu, cushion; De, dend oid; Fa, an; Le, lemnoid; M , ma ough;
Ms, ma smoo h; M , ma halloid; Sc, soli a y c eeping; S , soli a y
Table 1
Lis o species, amilies, o de s, and di isions appea ing in he selec ed pape s o
he me a-analy ical app oach. The numbe o imes each axon appea s among
he selec ed a icles is epo ed in he igh column.
Species (38) Families (19)
Ba bula inealis 1 Hypnaceae 6
B achy hecium saleb osum 1 Po iaceae 5
B yum cellula e 1 Thuidiaceae 4
Dic anum undula um 1 B achy heciaceae 3
Eu hynchium eus egium 1 Hylocomiaceae 3
Eu ohypnum lep o hallum 1 G immiaceae 2
Fon inalis an ipy e ica 1 Poly ichaceae 2
G immia anodon 1 Sema ophyllaceae 2
Haplocladium angus i olium 1 Aneu aceae 1
Haplocladium mic ophyllum 1 Ay oniaceae 1
Hylocomium splendens 1 B yaceae 1
Hypnum cup essi o me 1 Dic anaceae 1
Hypnum plumae o me 1 Fon inalaceae 1
Junge mannia exse i olia subsp. co di olia 1 Funa iaceae 1
Lunula ia c ucia a 1 Junge manniaceae 1
Ma chan ia polymo pha 1 Lejeuneaceae 1
Physcomi ella pa ens 1 Lunula iaceae 1
Plagiochasma appendicula um 1 Ma chan iaceae 1
Pleu ochae e squa osa 1 Sphagnaceae 1
Pleu ozium sch ebe i 1
Poly ichum commune 1 O de s (13)
Poly ichum pili e um 1 Hypnales 19
Pseudoscle opodium pu um 1 Po iales 5
P ilium c is a-cas ensis 1 G immiales 2
P ychan hus s ia us 1 Ma chan iales 2
Racomi ium lanuginosum 1 Poly ichales 2
Rhy idiadelphus squa osus 1 B yales 1
Ricca dia pinguis 1 Dic anales 1
Scopelophila ligula a 1 Funa iales 1
Sphagnum squa osum 1 Junge manniales 1
Taxiphyllum ba bie i 1 Lunula iales 1
Taxiphyllum axi ameum 1 Me zge iales 1
Taxi helium nepalense 1 Po ellales 1
Taxi helium sp. 1 Sphagnales 1
Thuidium cymbi olium 1
Thuidium delica ulum 1 Di isions (2)
Timmiella ba buloides 1 B yophy a 32
To ula u alis 1 Ma chan iophy a 6
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
4
halloid; T , u ; Th ead, h ead; Tp, u p o onemal; Ts, u sca e ed;
Tu , u ; We, we . Pe desc ibes he o m o pe enna ion wi h ou
ca ego ies: A, annual; AP, annual o pe ennial, mo e equen ly annual;
PA, pe ennial o annual, mo e equen ly pe ennial; P, pe ennial. Dis-
ibu ion o sex o gans is desc ibed by Sex ca ego ies: D, dioecious; D
(M), no mally dioecious, a ely monoecious; MD, monoecious o dioe-
cious; M(D), no mally monoecious, a ely dioecious; M, monoecious;
Nil, game angia unknown. Len alues epo in mm leng h o shoo o
hallus (o diame e o he ose e). L anges om 0 (plan s g owing in
da kness) o 9 (plan s g owing in ull ligh ). F goes om 1 (plan s ha
g ow in ex eme d yness) o 12 (no mally subme ged plan s). R anges
om 1 (g ow h condi ions o ex eme acidi y) o 9 (g ow h subs a a
wi h ee calcium ca bona e). N assumes alues be ween 1 (plan s
g owing in ex emely in e ile si es) and 7 ( ichly e ile g ow h condi-
ions). Finally, HM anges om 0 (no ole ance o HM) o 5 (plan s
con ined o subs a a p esen ing mode a e o a high concen a ion o
HM).
2.2.4. Fluo escence measu emen s
Fluo escence pa ame e s we e measu ed as a p oxy o i ness on
da k-adap ed plan s (30 min) a oom empe a u e using he imaging
luo ome e (Handy Fluo Cam FC 1000-H) and i s ela ed so wa e
Fluo Cam7 e sion 1.2.5.16. (P.S.I., B no, Czech Republic, h p://www.
psi.cz), as desc ibed in Es eban e al. (2008). Fluo escence was de ec ed
by a high-sensi i i y cha ge-coupled de ice came a equipped wi h a F
4.5–10 mm, 1:1.6 objec i e ha p oduced 12-bi , colou images. Images
o he da k-adap ed luo escence le el, Fo, we e de e mined using
non-ac inic measu ing lashes, ollowed by an 800 ms du a ion pulse o
sa u a ing ligh adia ion (2000
μ
mol pho on m−2 s −1) using 12
measu ing lashes o measu e he maximum luo escence le el, Fm. To
imp o e he signal- o-noise a io, bo h Fo and Fm we e a e aged. The
maximal pho ochemical e iciency o pho osys em II was es ima ed by
he a io F
V
/F
M
=(FM−F0)/F
M
. Da a we e collec ed in i e eplica es o
each o he ou sampled species and o p ocess he cap u ed images,
alse-colo images o lea F
V
/F
M
we e es ablished based on he
assump ion ha pixel in ensi y alues can be ela ed o he physiological
p ocess. In de ail, luo escence da a analysis was made in eg a ing e-
sul s o e he su ace o he pic u e o he moss samples on squa e a eas
o abou 200–350 pixels comp ising g een, pho osyn he ic apices o
ex ac he mos ele an in o ma ion.
2.2.5. Hea y me als analysis in moss issues
The d y samples we e weighed, pul e ized ( o ob ain a ep esen a-
i e sample) and hen subjec ed o acid diges ion o 15 min wi h 10 ml
o concen a ed HNO
3
a 180 ◦C in a mic owa e (CEM, MARS One). The
ob ained solu ion was hen dilu ed a 2%. The Cd, Pb and Zn concen-
a ions we e de e mined by induc i ely coupled plasma mass spec-
ome y (Agilen 7700), a e gene a ing calib a ion cu es o emission
using s anda d solu ions o each me al (Sigma).
2.2.6. Pho osyn he ic pigmen and ocophe ol con en analysis
Pigmen s and ocophe ols we e ex ac ed, analysed and quan i ied
ollowing he me hod o Es eban e al. (2014). Plan issues we e ho-
mogenized in pu e ace one cen i uged a 4 ◦C o 20 min a 16,000 ×g
he supe na an being sy inge- il e ed by a 0.22 µm PTFE il e
(Teknok oma, Ba celona, Spain). 15 µl o he ex ac o each eplica e
we e injec ed on a e e se-phase C18 column HPLC sys em (wa e
Sphe iso b ODS1, 4.6 ×250 mm, Mild o d, MA, USA) as epo ed in
Ga cía-Plazaola and Bece il (1999) and Fe n´
andez-Ma ín e al. (2018).
This s udy was ocused on compounds ha indica e he le el o pho o-
p o ec ion in b yophy es (Es eban e al., 2015). Fo his eason, in he
esul s, he ollowing compounds a e shown: he o al chlo ophyll pool
exp essed on a d y weigh basis (Chl a+b, nmol/g), he a o b chlo o-
phyll a io (Chl a/b, mol/mol), o al ca o enoids (sum o neoxan hin
(Neox), lu ein (Lu ), β-ca o ene (β-ca o ), Violaxan hin (V), An he ax-
an hin (A) and Zeaxan hin (Z)) exp essed on bo h DW (nmol/g) and
chlo ophyll basis (ca o enoids/Chl; mol/mol), o al xan hophyll pool on
DW (VAZ =V+A+Z; nmol/g), de-epoxida ion index (A+Z/V+A+Z;
mol/mol), Lu , Neox and β-ca o on DW basis (nmol/g). Finally,
α
- ocophe ol, γ +β- ocophe ols and o al ocophe ols (as he sum o he
isome s
α
, γ +β) we e also exp essed on a DW basis (nmol/g).
2.2.7. To al an ioxidan con en de e mina ion
The an i adical (an ioxidan ) ac i i y o moss samples (i.e., TAC),
based on he sca enging ac i i y o he s able ee adical 2,2-diphenyl-
2-pic ylhyd azyl (DPPH) was es ima ed acco ding o Cla ke e al.
(2013), wi h mino modi ica ions. Fi s , an ioxidan s we e ex ac ed
om mosses issues (50 mg) wi h 1.5 ml o a mix u e o me hanol:wa e :
ace ic acid (70:23:7). This sol en mainly ex ac s pola (hyd ophilic)
an ioxidan s such as asco ba e and phenols. Then, 50 µl o he ex ac
was added o 1.0 ml DPPH solu ion in me hanol (0.05 mM) and he
abso bance decay o he DPPH ee adical a 517 nm was de e mined
a e 60 min (in he da k) a oom empe a u e. The esul s we e
exp essed as asco ba e equi alen s as his an ioxidan was used as a
s anda d.
2.2.8. An hocyanin de e mina ion
An hocyanin de e mina ion was ca ied ou as desc ibed in Es eban
e al. (2008), analysing i e eplica es o each species om MP and NMP.
In de ail, app oxima ely 15 mg (FW) o plan ma e ial we e dis up ed in
liquid ni ogen and ex ac ed in 1 ml o 3 M HCl:H
2
O: MeOH (1:3:16 by
ol.) using a issue homogenise . Ex ac s we e cen i uged and an ho-
cyanin le els es ima ed as cyanidin-3-glycoside equi alen s using a
mola ex inc ion coe icien o 33,000 (Gould e al., 2000). The abso -
bance o an hocyanins a 524 nm was co ec ed by sub ac ing he
in e e ence by pheophy in as A524–0.24A653 (Mu ay and Hacke ,
1991).
Table 2
B yological a ibu es o he s udied species as conce ns: p ima y li e- o m (LF1), whe e T s ands o u , Cu o cushion and M o ough ma ; ype o pe enna ion
(Pe ), whe e P s ands o pe ennial; dis ibu ion o game angia in game opho e (Sex), whe e M s ands o monoecious and D o dioecious; game opho e’s size
exp essed in mm (Len); majo biome index (E1) alues, whe e biomes a e 3 wide bo eal, 5 bo eo- empe a e, and 7 empe a e biome; eas e n limi o dis ibu ion (E2)
index alues, whe e biogeog aphic elemen s a e 3 Eu opean and 6 ci cumpola ; Ellenbe g ligh alues (L) indica ing 4 shade o semi-shade plan s, 6 plan s g owing
om semi-shady o well-li ecological con ex s, 8 ligh -lo ing plan s, 9 plan s g owing in ull ligh ; Ellenbe g mois u e alues (F), desc ibing plan s g owing on 2
midway be ween ex eme d y and d y si es, 5 mode a ely mois soils, 6 mois soils o humid places, 7 cons an ly mois , no pe manen ly wa e logged subs a a;
eac ion o en i onmen al acidi y (R) alues indica e plan s g owing on 2 ex eme acid o acid subs a a, 5 mode a ely acid soils, 7 s ongly basic subs a a; ni ogen
alues (N) indica ing plan s g owing on si es 2 in e ile, 3 mode a ely in e ile, 4 mode a ely in e ile o mode a ely e ile, 6 mode a ely e ile o ichly e ile; and
ole ance o HM (HM ) alues indica ing 0 plan s absen om subs a es wi h mode a e o high concen a ions o HM and 1 plan s a ely eco ded on mode a e o high
concen a ions o HM.
Species LF1 Pe Sex Len (mm) E1 E2 L F R N HM
L. upes is Cu P M 40 5 3 8 2 5 3 0
P. commune T P D 250 3 6 6 7 2 2 1
P. compac um T P M 15 5 6 9 6 7 4 0
R. con e um M P M 30 7 3 4 5 7 6 0
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
5
2.2.9. pH measu emen s
In MP and NMP i e eplica es o each species (~10 mg DW) we e
selec ed o pH de e mina ion. This was ca ied ou as desc ibed in
Co nelissen e al. (2006). Plan ma e ial was mixed wi h 1 ml o
deionized wa e . The mix u es we e agi a ed a 250 pm o 1 h, hen
cen i uged a 16,000 ×g o 5 min. The pH o he luid was hen
measu ed using a na ow (5 mm diame e ) SenTix Mic elec ode con-
nec ed o an Inolab Le el 2 pH me e (bo h: WTW, Weilheim, Ge many).
2.3. S a is ical analysis
The no mal dis ibu ion o da a was assessed by he Shapi o-Wilk’s
es . Da a ep esen ing he pe cen age o a ia ion om con ol alues
(Fig. 1) we e es ed o signi ica i ely di e om a hypo he ical sample
o non-con amina ed b yophy es (NCB) wi h a one-sample - es o one-
sample Wilcoxon es , in case o no mally o non-no mally dis ibu ed
da a, espec i ely. In he expe imen al ield app oach, we c ea ed a new
ca ego y by a e aging samples in each ea men (he eina e All) ha
allows us o isualize whe he a a ia ion in a speci ic pa ame e due o
HM ( ep esen ed by boxplo s) is speci ic o each species o i i is a global
esponse. Wilcoxon es was used o de e mine i di e ences be ween
me allicolous and non-me allicolous popula ions (NMP and MP) we e
signi ican (Figs. 2–5). The p incipal componen analysis (PCA) o
analyse hea y me als composi ion and species ai s was pe o med
using he packages “Fac oMineR” and “ ac oex a”, o explain he a i-
abili y o da a in ela ion o he analysed a iables and o e alua e i
indi iduals clus e ed in ela ion o any o hem. All s a is ical analyses
we e ca ied wi h 1.2.1335 e sion o he R-S udio so wa e (R Co e
Team, 2019). Da a was conside ed signi ican a
α
=0.05 o below.
3. Resul s
3.1. Da a compiled om li e a u e e ealed a lack o ield s udies bu also
a global dec ease end o pho ochemical e iciency due o me als p esence
Jus 35 a icles esul ed sui able o be in eg a ed in o he analysis o
da a om he li e a u e in he p esen s udy (Table 1S). Among hem,
he g ea majo i y o pape s discussed esul s on indi iduals exposed o
HM s ess unde labo a o y condi ions. Only 5 pape s ( hose epo ed as
10, 21, 23, 26 and 32 in Table 1S) we e based on ield sampling and only
one a icle ( epo ed in Table 1S as 20) discussed da a analysed on in-
di iduals na u ally g owing in a pollu ed abandoned mining a ea. We
analysed he pe cen age o a ia ion be ween CB and NCB o se e al
ai s implica ed in pho op o ec i e esponse: Fluo escence pa ame e s
(F
V
/F
M
a io and NPQ; Fig. 1A), chlo ophylls (Chl a, Chl b, Chl a+b and
Chl a/b; Fig. 1B), ca o enoids (on weigh and chlo ophyll basis; Fig. 1C),
an hocyanins and asco ba e (Fig. 1D). We ound a 32% s a is ically
signi ican dec ease o F
V
/F
M
(Fig. 1A), oge he wi h a signi ican
dec ease in Chl a (36% o dec ease wi h espec o NCB), Chl a+b (38%
dec ease) and Chl a/b (10% dec ease) in CB as compa ed wi h NCB
mosses (Fig. 1B). Howe e , we did no ind a signi ican dec ease in Chl
b (Fig. 1B). Rega ding ca o enoids, no signi ican change was ound wi h
espec o CB (Fig. 1C). Al hough a 440% inc ease in he an hocyanins
con en and a 91% inc ease in hyd ophilic an ioxidan s as asco ba e
mani es ed in CB, hese da a esul ed o be no signi ican , p obably due
o he low numbe o eplica es (n =2–4) (Fig. 1D).
Fig. 1. Analysis o da a compila ion om he li e a u e. Boxplo s ep esen he
pe cen age o a ia ion o con amina ed b yophy es wi h espec o non-
con amina ed b yophy es (NCB) alues ( ep esen ed by he o ange dashed
line) o i) luo escence ela ed pa ame e s, i.e. pho ochemical e iciency (F
V
/
F
M
) and non-pho ochemical quenching (NPQ), (A), ii) chlo ophylls, i.e. chlo-
ophyll (Chl) a, b and Chl a+b (pe weigh uni ), and Chl a o b a io (Chl a/b)
(B); iii) o al ca o enoids, i.e. ca o enoids pe weigh uni and ca o enoids o
chlo ophyll basis (ca o enoids/Chl) (C); i ) an ioxidan s, i.e. an hocyanins and
asco ba e; pe weigh uni (D). Each boxplo epo s 50% o he measu ed
alues (inside he box), comp ised be ween he i s qua ile alue (lowe side
o he box) and he hi d qua ile alue (uppe side o he box), he median is
indica ed by he black line inside he box, while whiske s join he i s and hi d
qua iles wi h lowe and highe measu ed alue espec i ely (ou lie s a e e-
po ed by black do s). The numbe inside each box indica es he numbe o
eplica es. S a is ical signi icance was es ed by one-sample - es o Wilcoxon
es (in case o no mali y o non-no mali y o da a dis ibu ion, es ed by
Shapi o-Wilk’s es ) o pe cen age alues o a ia ion om con ol alues in he
selec ed pa ame e s and ep esen ed by as e isks when signi ican (*p <0.05;
**p <0.01; ***p <0.001). No e ha ns indica es non-s a is ical signi icance.
Fig. 2. Mean con en o hea y me als in moss issues (Cd, Pb and Zn;
μ
g/g) in
me allicolous and non-me allicolous popula ions (NMP and MP, ep esen ed by
g een and ed ba s espec i ely) o he ollowing species: L. upes is,
P. commune, P. compac um and R. con e um (n =3±S.E.). S a is ical signi i-
cance o di e ences be ween NMP and MP alues was es ed by - es and he
esul s a e ep esen ed by as e isks when signi ican (*p <0.05; **p <0.01;
***p <0.001). No e ha ns indica es non-s a is ical signi icance.
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
6
3.2. Expe imen al ield app oach highligh ed he species-speci ic ole ance
pa e ns
We analysed he me al composi ion (Cd, Pb and Zn) in bo h me al-
licolous (MP) and non-me allicolous (NMP) mosses. Zinc was he me al
ha eached he highes issue concen a ion in all he species, mos
likely due o he high a ailabili y le els o his me al p esen in soil (see
si e desc ip ion in ma e ial and me hods) as well as o i s high mobili y.
Besides, we ound species-speci ic me al con en (Fig. 2). In de ail, L.
upes is and R. con e um me allicolous popula ions p esen ed signi i-
ca i e highe alues o he h ee elemen s, whils P. compac um only
showed signi ica i e highe alues o he elemen Pb. On he o he
hand, P. commune seemed o a oid HM con en in issues wi h bo h
popula ions (MP and NMP) showing null (Cd) o e y low le els (Pb and
Zn).
Rega ding ai s induced by HM, we analysed he pho op o ec i e
esponses o he me allicolous and non-me allicolous popula ions o
mosses in his mining en i onmen . The pa e n obse ed o pho o-
chemical e iciency (Fig. 3) was simila o he esul s ob ained by he
analysis o da a compila ion om li e a u e, wi h signi ican ly lowe
alues o F
V
/F
M
o P. compac um and R. con e um g owing unde HM
(MP). Howe e , his dec ease (≈13%) was smalle han he dec ease
obse ed in he analysis o da a om he li e a u e. To be no iced, L.
upes is and P. commune did no show signi ican di e ences in F
V
/F
M
be ween bo h popula ions. I we a e age F
V
/F
M
esul s o he ou spe-
cies oge he , he s a is ically signi ican di e ence be ween NMP and
MP was main ained (Fig. 3, ca ego y All), indica ing a gene alized and
global dec ease end o his pa ame e unde HM p esence.
Rega ding pigmen s (Figs. 4–5), we ob ained species-speci ici y e-
sponses, howe e , any s a is ically signi ican di e ence be ween MP
and NMP was no main ained when he ou species alues we e a e -
aged (All boxplo s, Figs. 4–5). This means ha di e en ial esponses
be ween g ow h condi ions (MP and NMP) and species we e la e ed
and coun e balanced when plo ed oge he , due o opposi e pigmen
ends esponses in each species. In de ail, Chl a+b o MP signi ican ly
dec eased in P. commune, while in P. compac um inc eased and i was no
a ec ed in L. upes is (low o al chlo ophyll pool in bo h popula ions;
≈2000 nmol/g DW) and R. con e um (Fig. 4A). The a io Chl a/b
(Fig. 4B) dec eased signi ican ly only in wo species, L. upes is and
P. compac um. A signi ican inc ease in ca o enoids o chlo ophyll a io
(Fig. 4 C) was measu ed only o MP o P. commune. To al ca o enoids
(Fig. 4D) ollowed he same esponse obse ed in Chl a+b con en .
Analysing indi iduals’ ca o enoids con en (VAZ, AZ/VAZ, o al Lu ,
Neox, and β-ca o ; Fig. 5), we obse ed again ha he esponse end o
each MP moss was di e en , con i ming he lack o any s a is ically
signi ican di e ence be ween MP and NMP when alues p oceedings
om he ou species a e a e aged (All boxplo s, Fig. 5A-E). As expec ed,
L. upes is did no show any signi ican change in any o he ca o enoids
analysed, as was shown by he o al ca o enoid pool (Fig. 4 C, D). The
moss P. commune signi ican ly dec eased he β-ca o con en (Fig. 5E).
On he o he hand, P. compac um signi ican ly dec eased he de-
epoxida ion index in MP (Fig. 5B), oge he wi h a signi ican inc ease
o VAZ, Lu , Neox and β-ca o (Fig. 5A, C-E), whils R. con e um
exhibi ed a signi ican dec ease o o al xan hophyll pool, VAZ (Fig. 5 A)
Fig. 3. Maximum pho ochemical e iciency (F
V
/F
M
) in
me allicolous and non-me allicolous popula ions (NMP and
MP, ep esen ed by g een and ed ba s espec i ely) o he
ollowing species: L. upes is, P. commune, P. compac um
and R. con e um (n =5). The i s wo boxplo s (g ouped
in he ca ego y All and ligh blue-shaded) a e aged he
da a o he ou s udied species. Each boxplo epo s 50%
o he measu ed alues (inside he box), comp ised be-
ween he i s qua ile alue (lowe side o he box) and
he hi d qua ile alue (uppe side o he box), he median
is indica ed by he black line inside he box, while whiske s
join he i s and hi d qua iles wi h lowe and highe
measu ed alue espec i ely (ou lie s a e epo ed by black
do s). False colo s images om F
V
/F
M
a e shown o each
species, ep esen ed by he cold- o-ho colo g adien e-
po ed in he scale ba a he le . S a is ical signi icance o
di e ences be ween NMP and MP alues was es ed by -
es and he esul s a e ep esen ed by as e isks when sig-
ni ican (*p <0.05; **p <0.01; ***p <0.001). No e ha
ns indica es non-s a is ical signi icance.
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
7
bu an inc ease o he de-epoxida ion index (Fig. 5B) in MP.
We again ound a g ea di e en ial esponse on he accumula ion o
an ioxidan s be ween MP and NMP depending on he moss species
(Fig. 6). Ne e heless, when γ+β- ocophe ol alues we e conside ed
oge he (Fig. 6B, All boxplo s), hese compounds main ained a s a is-
ically signi ican inc ease in MP, meaning ha he same end is oughly
sha ed by each species conside ed singula ly as well. I we analyse he
esul s indi idually, we ound ha L. upes is was he only species
showing a signi ican inc ease o bo h
α
- ocophe ol and
γ+β- ocophe ol (Fig. 6A, B), bu wi h no changes in he es o he
pa ame e s. The MP o P. commune also inc eased signi ican ly
γ+β- ocophe ol, (Fig. 6B) and showed he highes basal alues o o al
an ioxidan capaci y (TAC) in bo h MP and NMP (Fig. 6C). The le el o
his hyd ophilic an ioxidan can be ela ed o e y low pH alues
measu ed in his species (Fig. 6E), especially in MP (in which pH alues
signi ican ly dec eased). MP o R. con e um inc eased signi ican ly
γ+β- ocophe ol (Fig. 5B) and an hocyanins (Fig. 5D). Besides,
R. con e um MP was he only case in which pH alues signi ican ly
inc eased (Fig. 6E). On he o he hand, MP o P. compac um did no show
any signi ican change in any an ioxidan wi h espec o he NMP
(Fig. 6A-D).
A p incipal componen analysis (PCA) was pe o med o help
disen angle species s a egies o cope wi h me allicolous g ow h con-
di ions (Fig. 7). The 53.4% o he o al a iance o he analysed da a was
explained by wo p incipal componen s. Mos o he o al a iance o he
PCA was explained by PC1, being he o al ca o enoids, Lu , Neox, Chl
a+b, VAZ and β-ca o he a iables ha be e explain PC1 ( ha alone
explains he 34.4% o he o al a iance o da a). The PC2, ha
explained 18.9% o he a iance, esul s o be e y in o ma i e as i
explains he in luence o Cd, Pb and Zn and main an ioxidan s (TAC,
γ+β- ocophe ol and
α
- ocophe ol). O e all, he PCA con i med he
end obse ed in p e ious Figs. (3–6). The MP and NMP o each species
clus e ed apa occupying di e en egions o he PCA space as a esul
o di e en and complex s a egies pu in place by he di e en species
o espond o he same ecological challenge. The species P. compac um
and R. con e um p esen ed highe con en o pollu an s and esul ed o
be hea ily in luenced in hei physiological pa ame e s in MP, while L.
upes is and P. commune p esen ed lowe /minimum con en s o pollu -
an s being less in luenced in hei physiological ai s.
4. Discussion
An h opocene ac i i ies and especially mining ac i i y caused local
and di use accumula ion o hea y me als ha migh h ea en he
Fig. 4. Boxplo s showing he o al chlo ophyll
con en (Chl a+b; nmol/g DW) (A); Chlo ophyll
a o b a io (Chl a/b, mol/mol) (B); ca o enoids
o Chl a io (ca o enoids/Chl; mol/mol) (C);
o al ca o enoids con en (D; nmol/g DW), in
me allicolous and non-me allicolous pop-
ula ions (NMP and MP, ep esen ed by g een
and ed ba s espec i ely) o he ollowing
species: L. upes is, P. commune, P. compac um
and R. con e um (n =5). The i s wo boxplo s
(g ouped in he ca ego y All and ligh blue-
shaded) a e aged he da a o he ou s udied
species. Each boxplo epo s 50% o he
measu ed alues (inside he box), comp ised
be ween he i s qua ile alue (lowe side o
he box) and he hi d qua ile alue (uppe side
o he box), he median is indica ed by he black
line inside he box, while whiske s join he i s
and hi d qua iles wi h lowe and highe
measu ed alue espec i ely (ou lie s a e e-
po ed by black do s). S a is ical signi icance o
di e ences be ween NMP and MP alues was
es ed by - es and he esul s a e ep esen ed
by as e isks when signi ican (*p <0.05;
**p <0.01; ***p <0.001). No e ha ns in-
dica es non-s a is ical signi icance.
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
8
en i onmen and human heal h (S ewa , 2019). Ongoing mining ac-
i i y is es ima ed on 57,277 km
2
o plane su ace (Maus e al., 2020),
howe e la ge a eas emain s ill a ec ed nowadays by he consequence
o pas and ceased mining ac i i ies. These habi a s wi h pas mining
ac i i ies his o y a e cha ac e ized by a combina ion o abio ic s esses
(e.g., high i adia ion, soil sca ci y, a idi y and HM-pollu ed g ow h
subs a e) and a e usually colonized by b yophy es ha adap o hese
me allicolous condi ions (due o hei ea ly land colonize s abili y; e.g.,
Holyoak and Lockha , 2011; G aham e al., 2014; So iano e al., 2019).
Ne e heless, he pho op o ec i e mechanisms in ol ed in his g oup o
plan s in he adap a ion and ole ance o HM pollu ed ecosys ems, such
as abandoned mining a eas, a e a o be deeply unde s ood. We
con i med his gap o knowledge by he analysis o da a compila ion
om li e a u e (Fig. 1, Table S1). Mos o he a icles consis ed indeed o
non-p eadap ed species o indi iduals suddenly exposed o HM ea -
men s unde labo a o y condi ions. Despi e he g ea con ibu ion ha
his app oach p o ided o he knowledge on b yophy es beha iou and
ole ance o HM s ess, his esul ed in add essing he ela ionship be-
ween b yophy es and HM wi h an acclima ion- ocused pe spec i e
a he han by a mo e adap i e-o ien ed iew. Indeed, among he 35
a icles selec ed, only 6 we e ocused on ield-sampled indi iduals. This
was he eason why we only ound h ee pa ame e s a ec ed in
con amina ed b yophy es: a dec easing end o pho ochemical e i-
ciency (Fig. 1) concomi an wi h o al chlo ophylls and chlo ophyll a o
b a io dec ease (Fig. 1B) unde a con amina ed en i onmen . This
esponse was due o chlo ophyll deg ada ion (Panda and Choudhu y,
2005; A es e al., 2018; Chen e al., 2018; Liang e al., 2018), an enna
size changes, mainly by chlo ophyll a deg ada ion (Shakya e al., 2008;
Aydo˘
gan e al., 2017; Nakajima and I oh, 2017); and p obably o pho-
oinhibi ion due o HM phy o oxici y exp essing a a chlo oplas le el
(Chen e al., 2015). Rega ding ca o enoids, we did no ind a consis en
esponse, possibly because di e en s a egies exis ed ega ding
HM-s ess esponse, and he o e all e ec s we e dilu ed (e.g., opposi e
pigmen s ends). This dilu ion e ec was also e iden when a e aging
he ou species da a in he expe imen al ield app oach (All boxplo ,
Figs. 3–6), indica ing high species-speci ici y and he possible ole o
di e en b yological a ibu es in he s udied species in de e mining he
a ie y o esponses o his s ess. Indeed, Co nelissen e al. (2007)
discussed how di e en c yp ogam ai s could ela e and in luence
ecosys ems’ p ope ies and highligh ed he c uciali y o deepening his
knowledge o unde s and impo an ecosys ems unc ions and so p edic
hei changes. Nowadays, he e is a lack o gene al ai pa e ns ha
could be applied o all b yophy e species globally. Mo e
HM- ole ance-o ien ed s udies o non- ascula plan s, oge he wi h a
Fig. 5. Boxplo s showing o al xan hophyll
con en (VAZ, nmol/ g DW) (A), deepoxida ion
a io (AZ/VAZ, mol/ mol) (B); lu ein con en
(Lu , nmol/ gDW) (C); neoxan hin con en
(Neox, nmol/ gDW) (D); β-ca o ene con en
(β-ca o , nmol/g DW) (E), in me allicolous and
non-me allicolous popula ions (NMP and MP,
ep esen ed by g een and ed ba s espec i ely)
o he ollowing species: L. upes is,
P. commune, P. compac um and R. con e um
(n =5). The i s wo boxplo s (g ouped in he
ca ego y All and ligh blue-shaded) a e aged
he da a o he ou s udied species. Each box-
plo epo s 50% o he measu ed alues (inside
he box), comp ised be ween he i s qua ile
alue (lowe side o he box) and he hi d
qua ile alue (uppe side o he box), he me-
dian is indica ed by he black line inside he
box, while whiske s join he i s and hi d
qua iles wi h lowe and highe measu ed alue
espec i ely (ou lie s a e epo ed by black
do s). S a is ical signi icance o di e ences be-
ween NMP and MP alues was es ed by - es
and he esul s a e ep esen ed by as e isks
when signi ican (*p <0.05; **p <0.01;
***p <0.001). No e ha ns indica es non-
s a is ical signi icance.
A. De Agos ini e al.
En i onmen al and Expe imen al Bo any 194 (2022) 104738
9
global scale me a-analysis conside ing species b yological a ibu es, a e
needed o e eal he pho op o ec i e esponses ac i a ed li ing in a
me allicolous en i onmen .
In he expe imen al ield app oach, we s udied b yophy es li ing in a
mine. The e o e, he esul s should be in e p e ed as adap i e mecha-
nisms g adually de eloped by b yophy es in hei s uggle o colonize
he me alli e ous niche and cope wi h he HM s ess and ela ed abio ic
s esses associa ed wi h hese en i onmen s. In gene al, we ound wo
impo an e en s: i) he me al con en ound in b yophy es esul ed o be
much highe han hose o ascula plan s g owing in he same s udy si e
(Ba u ia e al., 2009); ii) all he species analysed we e adap ed o g ow
and li e in he me allicolous si e. This was indica ed by he low educ-
ion o he pho osyn he ic e iciency be ween MP and NMP (Fig. 3),
which indica es down egula ion o he pho osys ems mo e han a pho-
oinhibi ion p ocess (Chen e al., 2015, 2018).
We also ound la ge adap i e in e speci ic di e ences be ween he
me allicolous b yophy e communi y (Fig. 8), wi h h ee main s a egies
ac i a ed o cope wi h he abio ic s esses p esen in he mining si e,
ou lined by he pho osyn he ic, pigmen s and an ioxidan esponses
oge he (Figs. 3–7) wi h he ecological a ibu es and g owing habi s o
he s udied species helping in explaining o he obse ed a iabili y
(Table 1):
i. Sensi i e s a egy depic ed by he species R. con e um. This
species is a pleu oca pous moss ha o ms ough, loose ma s
(Table 1, LF1 index), so his g ow h habi obliges he b yophy e o
li e in s ic ela ion wi h he pollu ed soil ha ing a g ea su ace
exposed o bo h we and d y a mosphe ic pollu an s deposi ion.
Pleu oca pous (p os a e) mosses seem mo e me al-sensi i e han
o he li e o ms as ac oca pous (e ec ) mosses (ˇ
Si ka e al., 2019).
This may explain he ac ha R. con e um was he species ha
accumula ed mos pollu an s in MP (Fig. 2) and he inc ease o i s
in e nal pH alues (Fig. 6E). We in e p e ed his as a consequence
o he basic ca ions’ up ake in he me alli e ous si e (a base- ich
en i onmen ) (Co nelissen e al., 2011), conside ing he in i-
ma e ela ion o he p os a e moss wi h he soil and i s elemen s
(ˇ
Si ka e al., 2019). All his caused a signi ican ly lowe pho o-
chemical e iciency in MP han in NMP (Fig. 3) indica ing ha
HM nega i ely a ec ed i s pho osyn he ic yield. To cope wi h his
pho osyn he ic ac i i y dec ease, he species pu in place he
pho op o ec i e mechanisms in MP ma ked by a signi ican
dec ease o o al xan hophyll pool, p obably due o ca o enoids
deg ada ion (Es eban e al., 2015), and a signi ican inc ease in
AZ/VAZ (Fig. 5 A and B), showing he ac i a ion o he iola-
xan hin cycle (Es eban e al., 2014). The pho op o ec i e s a egy
Fig. 6. Boxplo s showing
α
- ocophe ol con en
(nmol/g DW, A), γ+β- ocophe ol con en
(nmol/g DW, B); o al an ioxidan con en
(TAC, exp essed as asco ba e equi alen s; C);
an hocyanins, exp essed as cyanidin-3-
glycoside equi alen s con en (D); pH (E) in
me allicolous and non-me allicolous pop-
ula ions (NMP and MP, ep esen ed by g een
and ed ba s espec i ely) o he ollowing
species: L. upes is, P. commune, P. compac um
and R. con e um (n =5). The i s wo boxplo s
(g ouped in he ca ego y All and ligh blue-
shaded) a e aged he da a o he ou s udied
species. Each boxplo epo s 50% o he
measu ed alues (inside he box), comp ised
be ween he i s qua ile alue (lowe side o
he box) and he hi d qua ile alue (uppe side
o he box), he median is indica ed by he black
line inside he box, while whiske s join he i s
and hi d qua iles wi h lowe and highe
measu ed alue espec i ely (ou lie s a e e-
po ed by black do s). S a is ical signi icance o
di e ences be ween NMP and MP alues was
es ed by - es and he esul s a e ep esen ed
by as e isks when signi ican (*p <0.05;
**p <0.01; ***p <0.001). No e ha ns in-
dica es non-s a is ical signi icance.
A. De Agos ini e al.
