Beyond p oduc i i y: E ec s o ex eme
wea he e en s on ecosys em p ocesses
and bio ic in e ac ions
Disse a ion
zu E langung des akademischen G ades
D . e . na .
o geleg de
Fakul ä ü Biologie, Chemie und Geowissenscha en
de Uni e si ä Bay eu h
on
F au Julia Wal e (M.A.)
gebo en am 01.09.1981 in Memmingen
Die o liegende A bei wu de un e de Be euung on P o . Anke Jen sch in de Zei on
Mai 2008 bis Feb ua 2011 am Helmhol z Zen um ü Umwel o schung-UFZ in Leipzig,
und on Janua 2012 bis Ap il 2012 am Leh s uhl ü S ö ungsökologie an de Uni e si ä
Bay eu h ange e ig .
Volls ändige Abd uck de on de Fakul ä ü Biologie, Chemie und Geowissenscha en de
Uni e si ä Bay eu h genehmig en Disse a ion zu E langung des akademischen G ades eines
Dok o s de Na u wissenscha en (D . e . na .).
Disse a ion einge eich am: 11.04. 2012
Zulassung du ch die P ü ungskommission: 11.09.2012
Wissenscha liches Kolloquium: 25.10.2012
Am ie ende Dekanin: P o . D . Bea e Lohne
P ü ungsausschuss:
P o . Anke Jen sch (E s gu ach e in)
P o . Ch is iane We ne Pin o (Zwei gu ach e in)
P o . Michael Hauhs (Vo si zende )
P o . Thomas Foken
P o . John Tenhunen
Table o Con en s
1. Sho summa y o he hesis/ Ku ze Zusammen assung de Dok o a bei .......................................... 1
2. Backg ound o he hesis..................................................................................................................... 6
2.1. Clima e change and ex eme wea he e en s ............................................................................... 6
2.1.1. Tempe a u e ex emes .......................................................................................................... 8
2.1.2. P ecipi a ion ex emes .......................................................................................................... 8
2.2. Plan and ecosys em esponse owa ds ex eme wea he e en s................................................ 10
2.2.1. Mo phological and physiological esponse o single plan s o a ious clima ic s ess ypes . 11
Plan esponse o hea ............................................................................................................... 11
Plan esponse o os .............................................................................................................. 11
Plan esponse o d ough ......................................................................................................... 12
Plan esponse o hea y ain all................................................................................................ 12
2.2.2. Impac o ex eme wea he e en s on plan communi ies and ecosys ems......................... 13
Obse a ional s udies ............................................................................................................... 13
Expe imen al e idence on ex eme wea he e en s and plan communi ies ............................ 14
3. On his hesis..................................................................................................................................... 18
3.1. Objec i es o his hesis ............................................................................................................. 18
3.2. Ou line o manusc ip s............................................................................................................... 19
3.3. Eme ging esea ch ques ions ..................................................................................................... 21
3.3.1. Resilience and s ess memo y............................................................................................. 21
3.3.2. Ex eme wea he e en s and ecosys em p ocesses a mul iple le els................................. 22
3.3.3. Clima e change and land use .............................................................................................. 23
Lis o manusc ip s and decla a ion o own con ibu ion...................................................................... 24
P esen a ions o my wo k a con e ences.............................................................................................. 28
Cu iculum o he pos g adua e school HIGRADE ............................................................................. 29
Acknowledgemen s............................................................................................................................... 30
Re e ences o he In oduc ion .............................................................................................................. 31
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining ..................
p oduc i i y.................................................................................................................... 37
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance?....................................................................... 71
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes......................................................................................................................... 90
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough -memo y in g asses............... 105
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming,
and ex eme summe d ough ...................................................................................... 121
Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency .................................................................. 145
Manusc ip 7: Combined e ec s o mul i ac o clima e change and land-use on decomposi ion in
empe a e g assland ..................................................................................................... 167
Synopsis .............................................................................................................................................. 191
Sho summa y o he hesis
1
1. Sho summa y o he hesis/ Ku ze Zusammen assung de Dok o a bei
Unde global clima e change, ex eme wea he e en s, such as hea wa es, d ough o
hea y ain spells, a e p ojec ed o inc ease in magni ude and equency. As hese may a ec
ege a ion and ecosys ems mo e han g adual shi s in mean clima ic pa ame e s,
in es iga ing he consequences o ex eme wea he e en s ecen ly became an impo an issue
in clima e change esea ch. The main ocus o mos expe imen s in es iga ing e ec s o
ex eme wea he e en s on ege a ion is on p ima y p oduc i i y. In ou expe imen in
a i icially plan ed communi ies, e en an ex eme d ough o 1000-yea ecu ence did no
ha e e ec s on abo e- o below-g ound biomass p oduc ion om 2005-2010 (manusc ip 1).
Thus, he main objec i es o his hesis we e (1) o in es iga e i ex eme wea he
e en s ha e an e ec on ecosys em unc ions1 beyond p oduc i i y, (2) o es i such a high
esis ance o esilience2 in esponse o d ough ega ding p oduc i i y also exis s in mo e
na u ally g own plan communi ies and (3) o u he elucida e possible mechanisms o he
su p isingly la ge s abili y o he plan communi ies.
To in es iga e hese objec i es, se e al expe imen al s udies we e conduc ed in
a i icially plan ed, as well as in na u ally g own g assland communi ies and consequences o
ex eme wea he e en s o ecosys em p ocesses, such as decomposi ion and he bi o y we e
in es iga ed. In a po expe imen , i was s udied, i g ass plan s eac imp o ed owa ds
epea ed d ough when compa ed o a i s d ough and hus e eal a kind o d ough memo y.
Such a memo y migh be one possible, bu up un il now widely neglec ed mechanism o
esilience.
E en hough biomass p oduc ion emained s able in ou expe imen in a i icially
plan ed communi ies (manusc ip 1), biomass quali y was se e ely a ec ed by ex eme
d ough , he eby s ongly a ec ing he de elopmen o a he bi o e ca e pilla eeding on
d ough -exposed lea es (manusc ip 2). Fu he , plan compounds o he hos plan depended
on he composi ion o he plan communi y i was g own in. This in u n esul ed in s ong
e ec s on he la al mo ali y o he bi o es eeding on such plan s.
In con as o he s udy in a i icially plan ed communi ies (manusc ip 1),
abo eg ound ne p ima y p oduc i i y (ANPP) was educed in na u ally composed g assland
in esponse o ex eme ain all a iabili y, including an ex eme d ough ollowed by hea y
1 Ecosys em unc ions: P ocesses ha in ol e mo e han one ecosys em o ophic le el and a e impo an o he
main enance o he whole ecosys em (e.g. decomposi ion, which is impo an o nu ien u no e , o p o iding
ood o good quali y o sus ain ood webs)
2 Resilience is unde s ood he e as he ime equi ed o e u n o a s eady-s a e ollowing dis u bance (Holling
(1973); Gunde son (2000))
Sho summa y o he hesis
2
ain all (manusc ip 6). Fo age quali y was al e ed by d ough . Fu he mo e, mowing
equency s ongly al e ed o age quali y and biomass p oduc ion, bu did no in e ac wi h
ain all a iabili y and hus did nei he bu e , no ampli y e ec s o ex eme ain all
a iabili y. Despi e e ec s o ain all a iabili y on ANPP, g assland showed high esilience
a e d ough ollowed by hea y ain, as e ec s we e la ge sho ly a e he ex eme e en , bu
did no pe sis un il a second ha es la e in he yea .
In na u al g assland, ain all a iabili y and d ough also a ec ed ecosys em p ocesses,
he e li e decomposi ion, beyond p oduc i i y (manusc ip 7). D ough ollowed by hea y
ain pulses dec eased decomposi ion a es. Decomposi ion in mo e equen ly mown
meadows was mo e ulne able owa ds d ough exposu e. Win e wa ming and addi ional
win e ain had no long- e m e ec on decomposi ion. To conclude, p ojec ed inc eases in
d ough equency unde clima e change may inhibi decomposi ion and al e nu ien and
ca bon cycling along wi h soil quali y in empe a e g assland, whe eas a educ ion o snow
co e leading o mo e a iable soil su ace empe a u es may coun e ac inc eased
decomposi ion unde win e wa ming.
In his hesis, an ecological s ess memo y as one possible mechanism o esilience is
de ined as any esponse o a single plan a e a s ess expe ience ha imp o es he eac ion o
he plan owa ds u u e s ess expe ience and which is assessed on a whole plan le el
(manusc ip 3). This hesis u he p o ides e idence o a d ough memo y in g ass plan s
(manusc ip 4): Plan s epea edly subjec ed o d ough showed imp o ed pho o-p o ec ion and
a highe a e o li ing biomass when compa ed o plan s aced wi h hei i s d ough .
Simila ly, ee seedlings exposed o d ough in summe e ealed highe os esis ance du ing
win e , p o iding e idence o a long-las ing “c oss-s ess-memo y” (manusc ip 5).
To sum up, he hesis shows ha ex eme wea he e en s, e en hough nei he se e ely
a ec ing biomass p oduc ion in a i icially composed, no in na u ally g owing communi ies
in he long- e m, exe s ong in luence on physiological o biogeochemical pa ame e s, such
as plan compounds o soil bio ic ac i i y. These changes in u n modi y ecosys em unc ions
beyond p oduc i i y, o example he bi o y o decomposi ion, possibly al e ing bio ic
in e a ions and nu ien cycling. Fu he mo e, he indings imply ha plan s exhibi a s ess
memo y a e s ess exposu e, which may be one mechanisms leading o a high s abili y and
esilience upon equen s ess.

Ku ze Zusammen assung de Dok o a bei
3
Ku ze Zusammen assung de Dok o a bei
Im Zuge des globalen Klimawandels we den ex eme We e e eignisse, wie
Hi zewellen, Dü en ode S a k egene eignisse seh wah scheinlich häu ige und auch
in ensi e we den. Da diese Vege a ion und Ökosys eme s ä ke beein lussen können als
g aduelle Ände ungen in klima ischen Du chschni spa ame e n, is die Un e suchung de
Konsequenzen ex eme We e e eignisse in le z e Zei e s ä k in den Fokus de
Klimawandel o schung ge e en. Das Haup augenme k de meis en Expe imen e, die Folgen
ex eme We e e eignisse ü die Vege a ion un e suchen, lieg au de P imä p oduk i i ä .
Inne halb unse es Expe imen s in küns lich zusammengese z en Gemeinscha en wu de die
obe - und un e i dische Biomassep oduk ion du ch eine ex eme Dü e nich beein luss
(Manusk ip 1).
Dahe sind die Ziele diese A bei , zu un e suchen, (1) ob ex eme We e e eignisse
einen E ek au Ökosys em unk ionen1, auße de einen quan i a i en P oduk ion on
Biomasse haben, (2) ob die E gebnisse de hohen S abili ä in den küns lich
zusammengese z en A engemeinscha en auch ü die na ü lich gewachsenen
G ündlandbes änden gel en und (3) mögliche Mechanismen de e s aunlichen S abili ä de
P lanzengemeinscha en nähe zu beleuch en.
Da ü wu den meh e e Expe imen e in küns lichen und na ü lichen
P lanzengemeinscha en du chge üh , in denen Folgen ex eme We e e eignisse ü
Ökosys emp ozesse, wie z. B. S euabbau ode He bi o ie, un e such wu den. In einem
Top expe imen wu de auße dem un e such , ob G asp lanzen besse mi eine wiede hol en
Dü e im Ve gleich zu eine e s en Dü e umgehen können, und dami eine A
Dü egedäch nis au weisen. Ein solches Gedäch nis könn e ein mögliche , abe bishe wenig
e o sch e Mechanismus on Resilienz2 sein.
Obwohl die Biomassep oduk ion küns lich zusammengese z e Gemeinscha en s abil
blieb (Manusk ip 1), ände e sich die Biomassequali ä s a k du ch ex eme Dü e. Dadu ch
e ände e sich die En wicklung eine phy ophagen Raupe, wenn sie Blä e aß, die eine
Dü e ausgese z wo den wa en (Manusk ip 2). Auße dem beein luss e die
A enzusammense zung de Gemeinscha , in de die Fu e p lanze wuchs, die
P lanzeninhal ss o e, was die S e be a e de La en e ände e.
1 P ozesse, die meh als eine Ökosys emebene be e en und die ü die Au ech e hal ung des gesam en
Sys ems no wendig sind
2 Benö ig e Zei , um nach eine S ö ung wiede einen s abilen Zus and zu e eichen (Holling (1973); Gunde son
(2000))
Ku ze Zusammen assung de Dok o a bei
4
Im Gegensa z zu S udie in küns lich zusammengese z en Gemeinscha en
(Manusk ip 1) wu de die obe i dische Ne op imä p oduk ion (NPP) du ch den Ein luss on
ex eme Niede schlags a iabili ä , also ex eme Dü e ge olg on s a kem Regen, eduzie
(Manusk ip 6). Auch die Fu e quali ä wu de du ch die Dü e modi izie . Des Wei e en
beein luss e die Mahd equenz Fu e quali ä und Biomassep oduk ion. Alle dings konn e die
Mahd equenz die E ek e de ex emen Niede schlags a iabili ä wede abpu e n, noch
e s ä ken; es gab keine In e ak ion zwischen den beiden Fak o en. T o z de E ek e de
Niede schlags a iabili ä au die NPP zeig e sich wiede eine hohe Resilienz on G ünland
nach Dü e und S a k egen, da die nega i en E ek e di ek nach dem ex emen
We e e eignis seh s a k wa en, abe nich bis zu zwei en E n e Ende des Jah es anhiel en.
Auch im na ü lichen G ünland wu den Ökosys emp ozesse, hie S euabbau, neben
de P oduk i i ä beein luss : Dü e, ge olg on S a k egen, e inge e S euabbau a en.
S euabbau in ö e gemäh en Wiesen wu de du ch die Dü e s ä ke beein äch ig .
Win e e wä mung und zusä zlich applizie e Win e niede schlag ha en keine lang is igen
E ek e au den Abbau. Zusammen assend läss sich sagen, dass die o he gesag e Zunahme
on Dü en den S euabbau behinde n und dadu ch in Näh s o - und Kohlens o k eislau
eing ei en könn e. Das Tauen de Schneedecke bei Win e e wä mung üh e zu eine e höh en
Va iabili ä de Bodenobe lächen empe a u und könn e dami e höh en Abbau a en du ch
Win e e wä mung en gegen wi ken.
Die o liegende A bei de inie ökologisches S essgedäch nis als die S essan wo
eine Einzelp lanze, die die Reak ion diese gegenübe wiede hol em S ess e besse . Ein
solches S essgedäch nis könn e ein mögliche Mechanismus on Resilienz sein (Manusk ip
3). Die A bei zeig e s e Hinweise au ein Dü egedäch nis bei G assp lanzen. P lanzen, die
wiede hol e Dü e ausgese z wa en wiesen einen besse en Schu z o oxida i em S ess und
dadu ch meh lebende Biomasse au als P lanzen, die das e s e Mal eine Dü e ausgese z
wu den. Auch wa en Baumkeimlinge, die im Somme eine Dü e e uh en, im Win e
os esis en e , was au ein „C oss-S essgedäch nis“ hinweis (Manusk ip 5).
So zeig diese A bei , dass ex eme We e e eignisse, selbs wenn sie die
Biomassep oduk ion nich s a k ode lang is ig beein lussen, physiologische ode
biogeochemische Pa ame e , wie z. B. P lanzeninhal ss o e ode die Ak i i ä de
Boden auna, e ände n. Diese Ände ungen modi izie en wiede um Ökosys em unk ionen,
wie He bi o ie ode S euabbau, wodu ch mögliche weise lang is ig in bio ische
In e ak ionen ode S o k eisläu e eingeg i en wi d. Wei e hin leg diese A bei nahe, dass
Ku ze Zusammen assung de Dok o a bei
5
P lanzen, nachdem sie S ess ausgese z wa en, ein S essgedäch nis en wickeln können, das
zu e höh e S abili ä und Resilienz un e häu igen S esse eignissen üh .
Backg ound o he hesis
6
2. Backg ound o he hesis
2.1. Clima e change and ex eme wea he e en s
Ins umen al empe a u e eco ds show ha a wa ming o he clima e sys em o e he
las cen u y is unequi ocal (Hulme, 2005; Blenkinsop and Fowle , 2007; T enbe h e al.,
2007). The global mean su ace empe a u es ha e isen by 0.74 ° C (±0.18 ° C) on a e age
om 1905-2006 (Hulme, 2005; Blenkinsop and Fowle , 2007; T enbe h e al., 2007).
Wa ming was mos p onounced o e land egions, especially o e he no he n hemisphe e
du ing win e and sp ing (T enbe h e al., 2007). In Ge many empe a u es ha e isen by 1° C
om 1901-2000, wi h a mo e p onounced wa ming du ing win e (Schönwiese e al., 2005;
Zebisch e al., 2012). O he empe a u e indices, such as he global sea le el ise o a ound 17
cm in he las cen u y, he educ ion o snow co e in he no he n hemisphe e o he
widesp ead glacie e ea a e consis en wi h he eco d showing ising empe a u es
(T enbe h e al., 2007). Fu he mo e, global wa ming is accele a ing quickly: he wa ming
a e o 0.13° C pe decade om 1955-2005 is almos double he wa ming a e o 0.07 ° C pe
decade o 1906-2005 (Beie kuhnlein and Foken, 2008; T enbe h e al., 2007). I is now
widely acknowledged, ha an h opogenic g een house gas emissions accoun o he la ges
pa o obse ed wa ming since p eindus ial imes and ha he obse ed wa ming can no be
explained by in e nal o cing o na u al ex e nal adia i e o cing only (Hege l e al., 2007;
T enbe h e al., 2007).
Fu u e p ojec ions indica e a u he wa ming o be ween 1.1° C and 6.4 ° C un il
2100, depending on he emission scena io used in he model. E en i CO2 emissions we e
held cons an on he le el o he yea 2000 (which is al eady no ul illed), empe a u es
con inued o ise o a leas he i s hi d o he 21s cen u y (Meehl e al., 2007).
Along wi h ising empe a u es, o he componen s o he clima e sys em, o example
p ecipi a ion, a e obse ed and p ojec ed o change.
Modi ica ions in he magni ude, as well as in he equency and du a ion o ex eme
wea he e en s a e o inc easing conce n: Such changes may occu bo h h ough changes in
he mean o in he a iabili y o he dis ibu ion o a clima e a iable, causing
disp opo ionally la ge changes in he equency o in ensi y o wea he ex emes, compa ed
wi h he changes in he mean (Meehl e al., 2000b; Nicholls and Alexande , 2007) (Fig. 1).
Ex eme wea he e en s a e mo e and mo e esponsible o a la ge pa o clima e ela ed
damage o socie y and ecosys ems (Field e al., 2012).
Backg ound o he hesis
13
oxygen, o example by de eloping ai oo s. Plan s no adap ed o looding can some imes
acclima e by his ological modi ica ions, like ae enchymes. O he wise, hey expe ience
hypoxia o e en anoxia. This causes e men a ion ins ead o espi a ion in he oo cells,
which es ic s g ow h by a as deple ion o s o ed ca bohyd a es. Lac a e and e hanol
accumula e and migh cause cell damage by inc easing acidi y. A e e-ae a ion plan s migh
su e oxida i e damage by o ma ion o ROS (Schulze e al., 2005; Lambe s e al., 2008).
O en myko hiza a e damaged in hypoxic soils, which impai s he plan s nu ien supply.
2.2.2. Impac o ex eme wea he e en s on plan communi ies and ecosys ems
Obse a ional s udies
Besides physiological and mo phological al e a ions in single plan s, clima ic a iables a ec
species dis ibu ion and anges, phenological li e cycle e en s, communi y composi ion and
species in e ac ions (Hughes, 2000; Visse and Holleman, 2001; Wal he e al., 2002).
Many obse a ional s udies documen he e ec o he g adual wa ming on ege a ion:
Polewa ds o upwa ds ange shi s in esponse o wa ming ha e been obse ed o a ious
species, e.g. an upwa d shi o he eeline and o alpine plan s in Eu ope in he las decades
(Hughes, 2000; Wal he e al., 2002; Pa mesan and Yohe, 2003; Thuille , 2007). The ising
empe a u es also led o phenological shi s in many plan species, o ins ance o an ea lie
onse o bud bu s o lowe ing (Wal he e al., 2002). Wa me condi ions o en ma ch he
needs o in asi e plan s, ha can possibly es ablish mo e apidly and mo e widesp ead unde
new condi ions. An inc ease o he mophilic in asi e species has been documen ed in se e al
ecosys ems (Wal he e al., 2002). Clima e change may also lead o species ex inc ions, wi h
species in moun ain habi a s o he Medi e anean especially endange ed (McCa y, 2001;
Thomas e al., 2004; Thuille e al., 2005; Sch ö e e al., 2005).
Compa ed o obse a ions o he e ec s o g adual wa ming o plan communi ies,
popula ions and species dis ibu ion, obse a ional s udies in es iga ing he consequences o
ex eme wea he e en s a e a e, as he occu ence o ex eme clima ic e en s is also a e
(Meehl e al., 2000a; Gu schick and Bassi iRad, 2003; Jen sch e al., 2007). Rapid
ca as ophic shi s in communi y composi ion o en ollow dis u bances caused by ex eme
clima ic e en s (e.g. s o ms)(Sche e e al., 2001). E en less d ama ic e en s may cause
changes in species compe i i e and acili a i e in e ac ions (Be ness and Callaway, 1994;
Jen sch e al., 2007). Fo ins ance, compe i ion in ensi ies in plan -plan in e ac ions unde
ex eme d ough (Tielbo ge and Kadmon, 2000; Ludwig e al., 2004; Maes e and Co ina,
2004). Thus, na u ally occu ing d ough s cause long-las ing shi s in plan communi y

Backg ound o he hesis
14
composi ion (Allen and B eshea s, 1998; B eshea s e al., 2005; Muelle e al., 2005).
D ough u he educes o es esilience and p oduc i i y and is p ojec ed o inc ease ee
mo ali y (Thompson e al., 2009; Llo e e al., 2004; Noo me s e al., 2008; Allen e al.,
2010). Many ee-species in he Medi e anean a e p ojec ed o dec ease hei dis ibu ion due
o mo e se e e d ough s (Sch ö e e al., 2005). In mesic g assland, howe e , inc eased
p ecipi a ion a iabili y, leading o longe d y pe iods ollowed by mo e ex eme ain all
e en s, p omo ed plan coexis ence and hus s abilized di e si y (Adle e al., 2006).
The ex eme summe hea wa es in Cen al- and Wes e n Eu ope in 2003 and in
Eas e n Eu ope in 2010, accompanied by se e e d ough , caused c op ailu e and Eu ope-
wide educ ions in p ima y p oduc i i y (Ciais e al., 2005; Ba ioped o e al., 2011).
Wa m spells du ing win e ha e also been obse ed o cause damage, as hey may lead
o a loss o os acclima ion and hus inc eased damage upon ecu ing os . A win e “hea
wa e” in 2007 in no he n Scandina ia, accompanied by hawing, led o ex ensi e damage o
he dominan dwa -sh ubs (Bokho s e al., 2009). S imbeck e al. (1995) ound ha a
na u al haw du ing midwin e caused deha dening o mon ane ed sp uce. As global wa ming
ad ances he beginning o he g owing season, inc easing damage caused by la e os e en s
has been obse ed (Gu e al., 2008).
Expe imen al e idence on ex eme wea he e en s and plan communi ies
As obse a ional e idence on he impac s o ex eme wea he e en s is limi ed, se e al
con olled ield-expe imen s assessed e ec s o ex eme clima ic e en s on na u al o
a i icially composed ege a ion. The ad an ages o well-conduc ed expe imen s5 a e he
possibili y o inco po a e con ol ea men s and o minimize he in luence o con ounding
ac o s. Howe e , as such a educ ionis app oach implies a he a i icial condi ions a ely
ound in eali y, he ans e o expe imen al e idence on complex, na u al sys ems migh be
limi ed.
In he beginning o expe imen al clima e change esea ch (1990s), s udies es ing
e ec s o ex eme wea he e en s on plan communi ies we e sca ce (Jen sch e al., 2007;
manusc ip 1) and he majo i y o he expe imen s implemen ed changes in wea he ends,
such as wa ming o inc eased CO2. Un il 2006, esea ch in es iga ing he e ec s o ex eme
e en s accoun ed o only one i h o he expe imen al clima e change s udies published
5 Well-conduc ed expe imen s should include p ope con ol ea men s a ying only he ac o s udied, should
wo k wi h enough eplica es o ensu e s a is ical powe and should andomly assign ea men s and eplica es.
Fu he , ea men a i ac s and biases caused by he expe imen conduc o s ha e o be a oided (Hu lbe , 1984)
Backg ound o he hesis
15
(Jen sch e al., 2007). Mos expe imen s assessed abo eg ound p oduc i i y as main esponse
pa ame e and in es iga ed e ec s o d ough (manusc ip 1).
P ecipi a ion manipula ions:
Expe imen ally applied d ough dec eased g assland p oduc i i y in some s udies
(Mo ec o e al., 2004; an Ruij en and Be endse, 2010; de Boeck e al., 2011). Howe e ,
p oduc i i y was o en only a ec ed in esponse o d ough in a id habi a s (Gilgen and
Buchmann, 2009; Mi anda e al., 2009) o in gene ally d y yea s (Bloo e al., 2010). The
VULCAN expe imen s assessing da a a sh ubland si es ac oss Eu ope, also ound a end o
educed biomass p oduc ion a e d ough only a he d ie si es (Penuelas e al., 2004;
Penuelas e al., 2007). In mesic g assland, d ough o en had no long- e m e ec s on
p oduc i i y (Naud sa e al., 2011), which was also ound o he EVENT I expe imen
(manusc ip 1). Despi e o en no ha ing la ge e ec s on p oduc i i y, d ough al e s
belowg ound p ocesses, e.g. by educing soil espi a ion (EVENT I and CLIMOOR
expe imen : Emme e al., 2004; K eyling e al., 2008; Sowe by e al., 2008; Tobe man e al.,
2008; manusc ip 1).
Se e al s udies did no es he di ec e ec s o d ough , bu he e ec s o inc eased
ain all a iabili y ( ewe , bu la ge e en s, including long d y in e als and hea y ain
spells) on g assland pa ame e s. Some s udies showed a la ge e ec o mean annual
p ecipi a ion on p oduc i i y (Ba e e al., 2002; Chou e al., 2008), while o he s ound
ain all a iabili y o be a mo e impo an d i e o ANPP (Knapp e al., 2002; Fay e al.,
2003). In he Rain Manipula ion Plo s (RaMPs) expe imen a Konza P ai ie Biological
S a ion in Kansas, USA, a educ ion in soil espi a ion, plan CO2 up ake (Ha pe e al., 2005)
soil wa e con en (Fay e al., 2003) and p oduc i i y (Fay e al., 2003; Knapp e al., 2002) and
an inc ease in soil ni ogen a ailabili y and in plan di e si y (Knapp e al., 2002) was ound
in empe a e con inen al g assland unde inc eased ain all a iabili y (la ge bu ewe
ain all e en s wi h a cons an o e all ain all amoun (Heisle and Wel zin, 2006). Heisle -
Whi e e al. (2008, 2009) ound a dec ease in p oduc i i y a he empe a e pa o a ansec
and an inc ease in he semi-a id end unde ewe , bu la ge ain all e en s. In a Cali o nian
g assland, changes in p ecipi a ion pa e ns caused changes in ophic in e ac ions, e.g. a
educ ion in consume abundance on a longe ime scale ha o e ode di ec , au ecological
sho - e m e ec s (Su le e al., 2007).
The d ough s udies no applying compensa ing ain pulses show ha a id sys ems o
mesic sys ems in d y yea s a e mo e ulne able owa ds d ough . Thus, a su icien o e all
ain all amoun seems o be impo an o g assland eco e y, which was also ound in ou
Backg ound o he hesis
16
s udy wi hin he EVENT II expe imen (manusc ip 6). The impac o d ough in a id
ecosys ems seems o depend la gely on o e all ain all amoun o he occu ence o se e al
la ge ain pulses.
Expe imen s es ing e ec s o hea y ain all e en s on ege a ion a e a e. In he
EVENT I expe imen hea y ain all e en s had only mino e ec s on p oduc i i y (K eyling
e al., 2008).
Tempe a u e manipula ions:
Expe imen s applying no only g adual wa ming, bu ex eme hea pulses, a e sca ce.
A none e al. (2011) ound only sho - e med e ec s o an expe imen al hea wa e on he
p oduc i i y o he dominan g ass species in allg ass-p ai ie o Oklahoma, bu no changes in
mos o he s udied species. In cold biomes plan s pe o med be e du ing a wa ming pulse,
bu wo se a e wa ds, possibly due o a loss o cold esis ance and subsequen highe s ess
le els unde he ecu ing cold (Ma chand e al., 2005; Ma chand e al., 2006; Bokho s e al.,
2009), whe eas esh li e decomposi ion was una ec ed by wa ming pulses (Bokho s e al.,
2010). In he EVENT I expe imen , epea ed soil eeze- haw cycles caused an inc ease in
p oduc i i y o empe a e g assland (K eyling e al., 2010). Howe e , lagged s ess e ec s in
hea h communi ies diminished biomass wo ege a ion pe iods a e applying wa ming pulses
(K eyling e al., 2010).
Combined manipula ions o mul iple clima ic a iables:
Few expe imen s apply mul iple, combined clima ic s esso s on ege a ion:
The CLIMAITE p ojec (Mikkelsen e al., 2008) applying ele a ed CO2, d ough and
wa ming as single ac o s and in combina ion on sh ubland sys ems in Denma k ound mos ly
smalle esponses o nu ien cycling o he combined ea men s han o he single ea men s.
Ne e heless, he u u e clima e scena io combining all ac o s led o educed N u no e
(La sen e al., 2011). G ime e al. (2008) ound a la ge long- e m esis ance o in e ile,
es ablished g assland in esponse o wa ming, d ough s and wa e addi ions o e 13 yea s. A
mesocosm expe imen including he baceous species in Belgium also applied hea wa es and
d ough as single and combined ac o s (de Boeck e al., 2011; an Pee e al., 2004). They
ound ha nega i e e ec s o d ough on CO2 exchange, g ow h, su i al and biomass
p oduc ion we e exace ba ed by hea wa es, whe eas hea wa es alone had no e ec , due o
anspi a i e cooling.
The summa ized esul s demons a e ha in ensi ying d ough s migh educe
p oduc i i y and also ag icul u al yield, especially unde al eady d y condi ions, wi h smalle
Backg ound o he hesis
17
o no e ec s in mesic g assland. Fu he mo e, some s udies show ha ex eme e en s al e ne
ca bon balance and soil p ocesses, he eby al e ing nu i ional pa hways and soil quali y.
The e is an u gen need o u he combine mul iple clima ic s esso s, as e ec s o such
mul i ac o expe imen s migh poin in o ally di e en di ec ions as expec ed ou o he
esponse owa ds single ac o s (Mikkelsen e al., 2008). S udies in es iga ing pa ame e s
o he han p oduc i i y and soil espi a ion a e needed o elucida e e ec s on bio ic
in e ac ions and ecosys em p ocesses on mul iple le els.
On his hesis
18
3. On his hesis
3.1. Objec i es o his hesis
The p e ailing esponse pa ame e o mos expe imen s in es iga ing e ec s o
ex eme wea he e en s on ege a ion is p ima y p oduc i i y. In he EVENT I expe imen , in
which s a is ically ex eme wea he e en s we e applied on a i icially plan ed communi ies o
a ying species- and unc ional di e si y, he ex eme wea he e en s did su p isingly no
cause la ge and de imen al changes in g assland p oduc i i y (K eyling e al., 2010). The
applied ea men s could consequen ly no be called “ex eme clima ic e en s” sensu Smi h
(Smi h, 2011a), as, al hough being ex eme in hei magni ude and leng h ela i e o he
e e ence pe iod, hey did no cause an ex eme esponse o plan communi ies, such as
widesp ead species mo ali y o communi y b eakdown.
Howe e , al hough no se e ely a ec ing p oduc i i y, he wea he ea men s caused
mo e sub le changes on a physiological and biogeochemical le el ha a e summa ized in
manusc ip 1. Sligh changes, o example in plan me abolic compounds can a ec mul iple
ecosys em p ocesses and le els, o ins ance by dec easing pala abili y o he bi o es o by
changing decomposi ion a es, which in u n al e s ophic in e ac ions and nu ien cycling.
Thus, one objec i e o his hesis was o elucida e how ex eme wea he e en s a ec
ecosys em unc ions beyond p oduc i i y, such as plan -he bi o e in e ac ions o
decomposi ion.
Especially mesic g assland communi ies a e o en e y s able when aced wi h
ex eme d ough (see sec ion 2.2.2.), which was also shown in he EVENT I expe imen . Ye ,
he unde lying mechanisms o such a high s abili y a e no well unde s ood. Ano he objec i e
o his hesis is o u he elucida e possible mechanisms o he su p isingly la ge esis ance o
esilience o plan s and plan communi ies when aced wi h ex eme wea he e en s. He e, he
ocus is on a possible s ess memo y, as up un il now i is unclea , how plan s and plan
communi ies eac when s ess is applied epea edly o e a ela i ely sho ime span. On he
one hand, his migh lead o a s ep-wise educ ion in he abili y o eco e , un il a o al
b eakdown o he sys em (Sche e e al., 2001). On he o he hand, s ess acclima ion may
lead o a pe sis ing inc ease in s ess esis ance, a mechanisms ha could be ega ded as kind
o s ess memo y. The conside a ion o no only an inc eased e en magni ude, bu also o an
inc eased equency o e en s is u gen ly needed in s udying clima ic ex emes (Smi h,
2011b).

On his hesis
19
The EVENT I expe imen is highly con olled in e ms o species composi ion, as he
plan ed communi y composi ions we e kep cons an o e he yea s by pe iodically weeding.
To in es iga e i he indings o high s abili y in he a i icially composed plan communi ies
can be con e ed o mo e na u al sys ems, he EVENT II expe imen was es ablished on a
semi-na u al meadow in 2008. He e, no only ain all was manipula ed, bu also di e en
land-use scena ios we e implemen ed. This expe imen was also designed o answe he
ques ion whe he he e ec s o d ough o hea y ain a e caused by an o e all al e a ion in
mean annual ain all amoun , o by inc eased ain all a iabili y (la ge , bu ewe ain all
e en s) unde cons an annual ain all amoun . Fo his eason, in EVENT II ain all amoun
was kep cons an om 2009 onwa ds and only he size o and he in e als be ween he
ain all e en s we e a ied.
To sum up, he main objec i es o his hesis we e (1) o in es iga e i ex eme wea he
e en s ha e an e ec on ecosys em unc ions beyond p oduc i i y, (2) o es i he high
s abili y o esilience in esponse o d ough ega ding p oduc i i y also exis s in mo e
na u ally g own plan communi ies and (3) o u he elucida e possible mechanisms o he
su p isingly la ge esis ance o esilience o he plan communi ies.
3.2. Ou line o manusc ip s
The i s manusc ip summa izes 5 yea s o d ough esea ch in he a i icially plan ed
g assland communi ies o EVENT I. Ex eme d ough had no e ec on abo eg ound- o
belowg ound p oduc i i y. Ne e heless, se e al o he physiological and biogeochemical
pa ame e s we e a ec ed. I physiological changes on a lea le el in luence o he ecosys em
le els and p ocesses in he long- e m had hus o be in es iga ed.
The second manusc ip he e o e deals wi h changes in lea compounds caused by
ex eme d ough and esul ing e ec s on he bi o es eeding on such lea es. A second ocus o
his s udy was o elucida e e ec s o plan communi y composi ion on lea compounds and, as
a consequence, he bi o e de elopmen . The s udy showed ha changes in g ass compounds
caused by se e e d ough a ec ed he bi o es eeding on such g ass: Ca e pilla s ed wi h
d ough -subjec ed lea es showed signi ican ly highe su i al, a longe du a ion o la al
de elopmen and highe pupal weigh . Fu he , plan compounds o ou a ge g ass depended
on he composi ion o he plan communi y i was g own in, which in u n a ec ed he bi o e
de elopmen : La ae eeding on species- iches communi ies wi hou legumes showed he
highes mo ali y, which was closely linked o low p o ein con en in hese lea es. This s udy
p o ides e idence ha e en qui e sub le changes in plan s caused by d ough o communi y
composi ion a e able o in luence bio ic in e ac ions and may e en lead o desynch onisa ion
On his hesis
20
o ophic and phenological adjus men s unde clima e change. Fu he mo e, as clima e
change is likely o a ec plan communi y composi ion, his will u he a ec lea quali y
and hus plan -he bi o e in e ac ions.
The second objec i e o his hesis was o elucida e possible mechanisms o he high
s abili y o g assland p oduc i i y unde clima ic ex emes. In he i s h ee yea s o he
EVENT I expe imen , a d ough o 100-yea ecu ence was applied (leading o 32 days o
consecu i e d ough ), and in he nex yea s, a d ough o 1000 yea ecu ence was applied. In
e e y yea , he same plo s we e subjec ed o d ough . One possible mechanisms o esilience
migh be ha he communi ies buil up an ecological memo y ha helped hem o cope wi h
d ough in he ollowing yea s. As ecological memo y on a communi y le el is di icul o
assess, we ocused on an ecological s ess memo y on a single plan le el. Su p isingly ew
s udies in es iga ed i whole plan s a e able o emembe s ess and o eac imp o ed owa ds
a ecu en s ess e en . This issue is especially impo an as equency o ex eme wea he
e en s is p ojec ed o inc ease unde clima e change (Smi h, 2011b). Fu he , a common
de ini ion o s ess memo y o ecologis s is missing. The hi d manusc ip hus i s de ines
he concep o an ecological s ess memo y on a whole plan le el, e iews he ew exis ing
s udies indica ing s ess memo y a e clima ic s ess (d ough , os , hea ) and discusses
possible mechanisms o an ecological s ess memo y, including epigene ic ones.
A d ough memo y in g ass plan s was in es iga ed wi hin a po -expe imen in which
one g oup o plan s was subjec ed o a single d ough and he o he o ecu en d ough
(manusc ip 4). This s udy p o ided e idence ha g ass plan s a e able o emembe d ough
e en a e a ha es and esp ou ing and o show a highe pe cen age o li ing biomass, due o
imp o ed pho op o ec ion, when compa ed o plan s subjec ed o hei i s d ough . Simila ly,
he expe imen pe aining o manusc ip 5 es ed os ha diness o Pinus nig a ju eniles and
showed ha plan s exposed o d ough du ing summe e ealed highe os ha diness in
win e (manusc ip 5). As bo h, os and d ough s ess, in ol e dehyd a ion s ess, i migh
well be ha an ecological c oss-s ess memo y was in ol ed he e. Plan os ha diness in his
s udy was ela ed o a highe concen a ion o ca bohyd a es. Con en o ca bohyd a es is also
o en inc eased unde d ough (e.g. manusc ip 2). Thus, he c oss-s ess memo y indica ed in
manusc ip 5 migh be ela ed o he as e syn hesis o soluble ca bohyd a es.
To es i he indings o he a i icially composed plan communi ies also hold unde
mo e ealis ic condi ions, an ex eme d ough was also applied on na u ally g own g assland
communi ies in he EVENT II expe imen . He e, e ec s o inc eased ain all a iabili y
(changes in iming and dis ibu ion o ain all, bu no in o e all ain all sum) on he
On his hesis
21
p oduc i i y and some aspec s o o age quali y o es ablished g assland we e in es iga ed. In
con as o he indings in a i icially plan ed communi ies (manusc ip 1), ANPP and o age
quali y we e educed in na u ally composed g assland in esponse o ex eme d ough
ollowed by hea y ain all e en s (manusc ip 6). Mowing equency s ongly al e ed o age
quali y and biomass p oduc ion, bu did did nei he bu e , no ampli y e ec s o ex eme
ain all a iabili y on p oduc i iy, as i did no in e ac wi h ain all a iabili y manipula ions.
Despi e e ec s o ain all a iabili y on ANPP, g assland showed high esilience a e
ex eme sp ing d ough ollowed by hea y i iga ion, as e ec s we e la ge sho ly a e he
ex eme e en , bu did no pe sis un il a second ha es la e in he yea , when no di e ences
be ween he ain all a iabili y manipula ions appea ed. In he p eceding yea , when he
ex eme sp ing d ough was no ollowed by i iga ions and hus also ecei ed he smalles
o e all amoun o wa e , nega i e e ec s on p oduc i i y we e la ge and emained un il he
second ha es in la e summe . Then, o me ly d ough exposed communi ies s ill showed
educed biomass p oduc ion. This highligh s he impo an ole o a su icien o e all amoun
o ain all o eco e y p ocesses in empe a e g assland and is in acco dance wi h he d ough
s udies men ioned in sec ion 2.2.2., showing se e ely ad e se e ec s o d ough p ima ily in
d y yea s o in a id biomes. As his hesis in es iga es e ec s o ex eme wea he e en s on
ecosys ems beyond p oduc i i y, manusc ip 7 epo s indings o a long- e m decomposi ion
expe imen conduc ed wi hin EVENT II. Ex eme d ough educed li e decomposi ion when
li e bags we e exposed o d ough o six weeks wi hin an 11 mon h pe iod. Su p isingly,
low ain all a iabili y wi h egula i iga ion dec eased decomposi ion. Addi ional win e
ain accele a ed decomposi ion, whe eas win e wa ming had no e ec on decomposi ion, bu
educed snow co e and inc eased a iabili y o su ace empe a u es. Mo e equen mowing
s ongly s imula ed decomposi ion, which could be a ibu ed o changes in li e quali y.
Howe e , he s imula ing e ec o equen mowing was absen unde ex eme ain all
a iabili y including d ough . P ojec ed inc eases in d ough equency unde clima e change
may inhibi decomposi ion and al e nu ien and ca bon cycling along wi h soil quali y.
Especially decomposi ion in in ensi ely managed g assland appea s ulne able owa ds
d ough .
3.3. Eme ging esea ch ques ions
3.3.1. Resilience and s ess memo y
O en, and also in ou s udy (manusc ip 1) g assland shows a su p isingly la ge
esis ance o esilience owa ds d ough . Mechanisms o esilience emain o be elucida ed.
On his hesis
22
One likely mechanism is a s ess memo y o plan s ha ende s hem less ulne able o
epea ed s ess e en s (manusc ip s 3, 4, 5). Howe e , i such a mechanisms exis s also unde
na u al condi ions and also on la ge scales, e.g. on a communi y le el, is ye o be
in es iga ed, especially as indings o manusc ip 6 imply ha g assland esilience unde mo e
na u al condi ions migh be diminished unde gene ally d y condi ions. Possible mechanisms
o a s ess memo y a e la gely unknown. Join esea ch o ecologis s and molecula biologis
is needed o elucida e possible epigene ic mechanisms. Fi s s udies al eady showed he
he i abili y o acqui ed s ess ole ance (see manusc ip 3). Besides ecological s ess memo y,
o he unde lying physiological and biogeochemical p ocesses ha se e o main ain
p oduc i i y and migh hus be mechanisms o communi y s abili y and eco e y ha e o be
iden i ied and add essed in u u e esea ch. Main aining ecosys em esilience is o majo
impo ance o mi iga e and p e en ca as ophic consequences o global clima e change.
3.3.2. Ex eme wea he e en s and ecosys em p ocesses a mul iple le els
Up un il now, he main esponse pa ame e s udied in esea ch on ex eme clima ic
e en s is p ima y p oduc ion (manusc ip 1). Howe e , e en i p ima y p oduc ion emains
s able, o he physiological and biogeochemical pa ame e s a e changed unde ex eme
wea he e en s (manusc ip s 1, 2, 7). Such changes, e.g. ood plan quali y migh se iously
in e e e in ecosys em synch onisa ion and ecosys em unc ioning. Fu he wo k o s udy
long- e m e ec s o ex eme wea he e en s on, e.g. bio ic in e ac ions o biodi e si y is
needed o es ima e consequences o wea he ex emes and o enable policy make s o p e en
des abiliza ion o es ablished ood-webs and o seize measu es o adap a ion. How he bi o es
migh eac o changes in hei hos plan in mo e na u al condi ions han he ones desc ibed in
manusc ip 2 and whe he specialis s migh eac di e en ly compa ed o gene alis
he bi o es also needs u he esea ch. We showed ha win e wa ming did no inc ease
decomposi ion, due o loss o snow insula ion and inc eased su ace empe a u e a iabili y.
How decomposi ion migh be a ec ed by summe wa ming, also in combina ion wi h d ough
condi ions, needs u he s udy. Long- e m changes in soil bio ic ac i i y unde mo e equen
mowing needs o be add essed, o ind explana ions o he highe ulne abili y o
decomposi ion owa ds d ough in mo e equen ly mown communi ies. P elimina y esul s o
he EVENT expe imen s also indica e s ong e ec s o hea y ain all on bio ic in e ac ions,
such as myco hiza o decompose auna. As hea y ain e en s a e expec ed o inc ease in he
u u e, bu a e a ely s udies ye , mo e in es iga ions a e needed o look a e ec s o hea y
ain on ecosys em unc ions.
Cu iculum o pos g adua e school
29
Cu iculum and c edi poin s o he pos g adua e school HIGRADE and
awa d
cou se name du a ion own con ibu ion c edi poin s
In oduc ion o wa e esou ces and aqua ic
ecosys em managemen
3 days ac i e pa icipa ion
and homewo k
1
In oduc ion in o biodi e si y sciences 2 days ac i e pa icipa ion 1
Ad anced cou se e es ial ecosys em
unc ions and biodi e si y
3 days ac i e pa icipa ion 2
Ad anced cou se p o eomics 5 days lab wo k and analysis 2
Semina on land-use con lic s and conse a ion
o na u al esou ces
1 day p esen a ion and
ac i e pa icipa ion
1
Applica ion cou se “Land-use con lic s and
conse a ion o na u al esou ces in he
Banaue egion o No he n-Luzon/ Philippines
12 days conduc ion o ield
expe imen and w i ing
o inal epo
3
So Skills: P esen a ions in Englisch 2 days ac i e pa icipa ion
including sho
p esen a ions
1
So Skills: Scien i ic W i ing 3 days ac i e pa icipia ion
including w i ing o
sho sec ions
1
So Skills: G an Aquisi ion 1 day pa icipa ion 0.25
Fou alks a in e na ional con e ences p epa a ion and
p esen a ion o alks
2
Fou p esen a ions in he UFZ  epa a io
semina and one pos e p esen a ion a he
UFZ Topic I con e ence
p epa a ion and
p esen a ion o alks
1.5
O ganisa ion o UFZ doc days 2009 se e al days planning o loca ion,
ac i i ies, alks,
schedule
0.75
S a is ics: Da a Analysis and Modelling using R 6 days ac i e pa icipa ion 1.25
Publica ion o a icles in ISI-lis ed jou nals p epa a ion o
manusc ip s, i s and
co esponding au ho
2
Pa icipa ion a he compe i ion “Wissenscha
Ve s ehen” and
AWARD o he 3 d place
Finals we e
held one day
 epa a ion o a icle
and 15 minu e
p esen a ion
1
20.75

Acknowledgemen s
30
Acknowledgemen s:
I hank
- P o . Anke Jen sch o gi ing me he oppo uni y o do my phD wi hin he g oup, o gi ing
me space o de elop own in e es s and ideas, o p o iding a wo king a mosphe e ha makes
ha d wo k easie , o gi ing me he chance o p esen my wo k a con e ences and o being
he amily- iendlies supe iso
- my second supe iso , D . Ha ald Auge, o many ui ul discussions and ideas o my
esea ch
- he depa men o Conse a ion Biology a he Helmhol z-Cen e o En i onmen al
Resea ch o my nice “win e -home”, D . Klaus Henle o gi ing me he oppo uni y o wo k
in Bay eu h du ing summe and he g adua e school HIGRADE o a lo o in e es ing cou ses
and ainings and o unding some esea ch s ays
- P o . Ca l Beie kuhnlein o gi ing me he possibili y o use (wo)man-powe and echnical
esou ces o his g oup and o as and help ul edi ing o my manusc ip s
- D . G ego Aas and he s a o he Ecological Bo anical Ga den o he Uni e si y o
Bay eu h o hei suppo o he whole expe imen
- P o . Wol am Beyschlag (Uni e si y o Biele eld), Jun.P o . Ch is iane We ne -Pin o
(Uni e si y o Biele eld), P o . John Tenhunen (Uni e si y o Bay eu h), D . Uwe Rasche
(FZ Jülich) and Ha ald Auge (UFZ Halle) o p o iding us hei echnical equipmen and D .
Sonja Lö le (LFE Ebe swalde) o he help and suppo wi h he lea chemical analysis
- nume ous s uden wo ke s and in e ns wi hin he EVENT- expe imen s o hei help in
main enance, ea men execu ion and o doing measu emen s wi h me, e en “p e-dawn”,
especially o Roman Hein, Lau a Nagy, Inés Pas o , Julia Gommola, Jan Taucha, Julia Smi h,
Da id Eichenbe g and Clesio Gomes da Sil a
- all he echnicians o hei gene al help in he expe imen , and especially Ch is ian Schemm
and Ch is ine Pilsl o hei help in he lab and Reinhold S ahlmann o his help wi h
“compu e -s u ”
- all my co-phDs (Ke s in, Lau a, Da id, Roman, Daniel, Jan) and ou pos -doc, Jü gen
K eyling, o making ield wo k un, o physical and psychological suppo and o many
use ul discussions
- my pa en s, sis e s iends and Roman and Smilla (I hope hey all know wha o )
Re e ences
31
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Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
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Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions
while main aining p oduc i i y
Jou nal o Ecology, 2011, 99: 689–702.
Anke Jen sch1*, Jue gen K eyling2, Michael Elme 3, Ellen Gellesch2, B uno Glase 4, Ke s in
G an 2, Roman Hein2, Ma co La a4, Heyda Mi zae6, S e anie E. Nadle 2, Lau a Nagy1, Denis
O ieno6, Ka in P i sch7, Uwe Rasche 8, Ma in Schädle 9, Michael Schlo e 7, B ajesh K.
Singh10, Ju a S adle 9, Julia Wal e 11, Camilla Wells ein2, Jens Wöllecke3 and Ca l
Beie kuhnlein2
1 Dis u bance Ecology, Uni e si y o Bay eu h, D-95440 Bay eu h, Ge many. E-mail: anke.jen sch@uni-
bay eu h.de
2 Biogeog aphy, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many.
3 Soil P o ec ion and Recul i a ion, BTU Co bus, Kon ad-Wachsmann-Allee 6, 03046 Co bus, Ge many.
4 Soilphysics, Uni e si y o Bay eu h, 95449 Bay eu h, Ge many
5 Soil Biogeochemis y, Ma in-Lu he Uni e si y o Halle-Wi enbe g, on Seckendo pla z 3, 06120 Halle,
Ge many.
6 Plan Ecology, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many.
7 Te es ial Ecogene ics, Helmhol z Zen um Muenchen, Ge man Resea ch Cen e o En i onmen al Heal h,
Ingols aed e Lands aβe 1, 85764 Neuhe be g, Ge many.
8 Ins i u e o Chemis y and Dynamics o he Geosphe e, ICG-3, Phy osphe e, Resea ch Cen e Jülich, Leo-
B and -S ., D-52425 Jülich, Ge many.
9 Communi y Ecology, Helmhol z-Cen e o En i onmen al Resea ch – UFZ, Theodo -Liese -S . 4, 06110
Halle, Ge many.
10 Cen e o Plan s and En i onmen , Uni e si y o Wes e n Sydney, Pen i h Sou h DC, NSW, Aus alia.
11 Conse a ion Biology, UFZ-Helmhol z, Cen e ü En i onmen al Resea ch, Pe mose s . 15, 04318 Leipzig,
Ge many.
Co esponding au ho : P o . D . Anke Jen sch, E-mail: [email p o ec ed]e
Running i le: D ough e ec on mul iple ecosys em se ices
Summa y
1. S udying he e ec s o ex eme clima ic o wea he e en s such as d ough and hea wa es
on biodi e si y and ecosys em unc ions is one o he mos impo an ace s o clima e change
esea ch. In pa icula , p ima y p oduc ion is amoun ing o he common cu ency in ield
expe imen s wo ldwide. Ra ely, howe e , a e mul iple ecosys em unc ions measu ed in a
single s udy in o de o add ess gene al pa e ns ac oss di e en ca ego ies o esponses and o
analyse e ec s o clima e ex emes on a ious ecosys em unc ions.
2. We se up a long- e m ield expe imen , whe e we applied ecu en se e e d ough e en s
annually o i e consecu i e yea s o cons uc ed g assland communi ies in cen al Eu ope.
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
38
The 32 esponse pa ame e s s udied we e closely ela ed o ecosys em unc ions such as
p ima y p oduc ion, nu ien cycling, ca bon ixa ion, wa e egula ion and communi y
s abili y.
3. Su p isingly, in he ace o se e e d ough , abo e- and below-g ound p ima y p oduc ion o
plan s emained s able ac oss all yea s o he d ough manipula ion.
4. Ye , se e e d ough signi ican ly educed below-g ound pe o mance o mic obes in soil
indica ed by educed soil espi a ion, mic obial biomass and cellulose decomposi ion a es as
well as myco hiza ion a es. Fu he mo e, d ough educed lea wa e po en ial, lea gas
exchange and lea p o ein con en , while inc easing maximum up ake capaci y, lea ca bon
iso ope signa u e and lea ca bohyd a e con en . Wi h ega d o communi y s abili y, d ough
induced complemen a y plan –plan in e ac ions and shi s in lowe phenology, and
dec eased in asibili y o plan communi ies and p ima y consume abundance.
5. Syn hesis. Ou esul s p o ide he i s ield-based expe imen al e idence ha clima e
ex emes ini ia e plan physiological p ocesses, which may se e o egula e ecosys em
p oduc i i y. A po en ial eason o di e en dynamics in a ious ecosys em se ices acing
ex eme clima ic e en s may lie in he empo al hie a chy o pa e ns o as e sus slow
esponse Such da a on mul iple esponse pa ame e s wi hin clima e change expe imen s os e
he unde s anding o mechanisms o esilience, o syne gisms o decoupling o
biogeochemical p ocesses, and o undamen al esponse dynamics o d ough a he ecosys em
le el including po en ial ipping poin s and h esholds o egime shi . Fu u e wo k is needed
o elucida e he ole o biodi e si y and o bio ic in e ac ions in modula ing ecosys em
esponse o ex eme clima ic e en s.
Keywo ds: below-g ound, compe i ion, decomposi ion, in asion, lea chemis y, mic obial,
phenology, plan –clima e in e ac ions, p ecipi a ion change, p oduc i i y
In oduc ion
Cu en ly, knowledge abou ecological esponses o clima e change is based la gely on
e ec s o clima ic ends such as g adual wa ming, p ecipi a ion change and CO2 en ichmen .
Howe e , he magni ude and equency o ex eme clima ic o wea he e en s such as se e e
d ough , hea wa es, hea y ain and la e os e en s a e expec ed o inc ease in he nea
u u e (IPCC 2007; O’Go man & Schneide 2009). Thus, p edic ions o e ec s o clima e
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
45
d ough . The maximum quan um e iciency o pho osys em II was calcula ed as F /Fm.
Va iable luo escence (F ) and maximum luo escence (Fm) we e measu ed be o e dawn.
Va iable luo escence was calcula ed as Fm-F0, Fm being he maximum luo escence o he
da k-adap ed lea a e applying a sa u a ing ligh pulse and F0 being he s eady-s a e
luo escence yield o he da k-adap ed lea (Maxwell & Johnson 2000). To enable a
compa ison be ween absolu e luo escence alues, a luo escence s anda d ma e ial was
measu ed be o e dawn and calcula ed as F /Fm (F = Fm – F0) (Maxwell & Johnson 2000).
Absolu e F0 and Fm alues we e aken o sepa a e he e ec s o pho odamage, becoming
appa en wi h an inc ease o F0, om he e ec s o pho op o ec ion ela ed o enhanced non-
pho ochemical quenching, becoming appa en wi h a dec ease in Fm (Wal e e al., 2011).
Lea gas exchange
Ca bon dioxide assimila ion (A) a he lea was moni o ed in A. ela ius in all he
g assland communi ies. (No da a could be ob ained om H. lana us in he pa icula yea o
da a mining due o i s lea e s a us.) A se ies o weekly measu emen s we e ca ied ou using a
po able gas exchange sys em (LI-6400, LI-Co , Lincoln, NE, USA). A se o 3 g ass u s on
each plo we e iden i ied and ma ked o measu emen s. On any measu emen day, 2-3
sui able lea blades selec ed om each o he u s pe plo we e se pa allel in he cu e e,
wi h hei uppe su aces well exposed so ha hey we e ully illumina ed du ing
measu emen s. E e y u n o measu emen s las ed one o wo minu es, when a s eady s a e
was a ained and a se o 10 eadings pe measu emen logged a 10-s in e als. The selec ed
lea es we e ma ked and simila lea es we e moni o ed ei he du ing midday (12:00 o 14:00
h) o h oughou he day ( om sun ise o sunse ), when diu nal cou se measu emen s we e
conduc ed. The measu ed lea es we e hen excised a he end o he measu emen pe iod and
he lea a ea (LA) o he sec ion o lea enclosed in he cu e e de e mined using lea a ea
me e CI-202 CID, Camas, WA, USA. Lea a ea in o ma ion was hen used o s anda dize he
lea gas exchange da a.
Soil espi a ion
In si u a es o soil espi a ion we e measu ed using a po able CO2 in a ed gas
analyse (EGM-4, PP Sys ems, Amesbu y, USA) linked o a soil espi a ion chambe (SRC-1,
PP Sys em, Amesbu y, USA). A he beginning o he ege a ion pe iod, pe manen PVC
colla s (10 cm diame e , 5 cm heigh , ligh g ey colou ) we e ins alled in e e y plo wi h a 1-
cm edge abo e soil su ace o ealize a closed sys em when he soil espi a ion chambe was
placed on he colla du ing measu emen . The day be o e each measu emen , all abo e-
g ound ege a ion was emo ed om he colla using scisso s. Du ing he ime ame o 8:00

Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
46
o 12:00, he soil espi a ion chambe was placed o 240 seconds on he colla o e e y plo .
An in e nal an ealized he e en dis ibu ion o ai and he in a ed gas analyse moni o ed
he build-up o CO2 wi hin he sys em. The a es o soil espi a ion we e de e mined om his
by i ing a quad a ic equa ion o he change in CO2 concen a ion wi h ime. Fo his s udy,
we analysed he soil espi a ion a es a second 240 o each high-di e si y g assland plo
including A. ela ius, H. lana us, P. lanceola a and G. p a ense on he las day o d ough
manipula ion.
Maximum lea and canopy up ake a es
Ne ecosys em CO2 exchange was measu ed wi h chambe s on 40 × 40 cm ames
es ablished on each o he ea men plo s. Daily cou se o ne ecosys em CO2 exchange
(NEE) was measu ed using manually ope a ed, closed gas exchange canopy chambe s. Ligh -
esponse cu es depic ing he ne pho osyn he ic CO2 up ake a e (A) o plan s a any
measu ing ime we e ob ained om lea -le el gas-exchange measu emen s by i ing an
empi ical ec angula hype bola model (Gilmano e al. 2005): NEE = (α+Q / αQ-β) - γ,
whe e α is he ini ial slope o he ligh - esponse cu e and an app oxima ion o he canopy
ligh u iliza ion e iciency (mol CO2/ mol PAR), β is he maximum CO2 up ake capaci y
(µmol m–2 s–1), Q is he pho osyn he ically ac i e adia ion (PAR, in µmol m–2 s–1), and γ is an
app oxima ion o he a e age day ime ecosys em espi a ion (lmol m–2 s
–1). An
app oxima ion o maximum canopy up ake capaci y was ex apola ed om lea -le el
measu emen s. Canopy ne ecosys em exchange a e (NEE) was es ima ed om lea
pho osyn he ic a e a sa u a ing ligh in ensi ies (i was shown ha A a PAR = 2000 µmol m–
2 s–1 co ela es well wi h canopy NEE). Maximum g oss p ima y p oduc i i y (GPPmax) was
calcula ed as: GPPmax = NEE2000 – Reco, whe e A2000 is he maximum lea pho osyn he ic a e
a a sa u a ing le el o ligh in ensi y andReco is he co ec ed espi a ion e m (γ) ob ained
om he model.
Nu ien cycling
In si u decomposi ion a e o cellulose
Biological ac i i y o soil auna and mic oo ganisms was de e mined indi ec ly om
he decay o cellulose using mini-con aine ubes (K eyling e al. 2008a). In o al, 864 mini-
con aine s we e illed wi h 0.2 g o cellulose (poo in phospho us, Schleiche & Schüll,
Dassel, Ge many) each, closed wi h a 2-mm mesh, and pu in o con aine ubes, consis ing o
12 mini-con aine s each. Two ubes we e bu ied ho izon ally 1 cm below soil su ace in each
g assland plo . A e 94 days, one ube pe plo was ha es ed, whe eas he o he s we e
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
47
ha es ed a e 186 days. A e ca e ul cleaning and d ying, he decay o cellulose was
de e mined by sub ac ing inal ashes- ee d y mass om ini ial d y mass (105 °C).
Myco hizal coloniza ion
One comple e plan indi idual o P.lanceola a was aken om each plo on he las
day o d ough using a soil co e sample wi h 5 cm diame e (Eijkelkamp; Ne he lands). This
pa icula species was chosen, because p e-analysis e ealed highe e ec s o d ough on
myco hizaal coloniza ion o P. lanceola a han on ha o o he species es ed. Roo s we e cu
o and ixed in o malin-alcoholic-acid (50 % E hanol, 40 % H2O, 7.5 % o malin, 2.5 %
acidic acid), and s ained wi h 5 % blue ink inega solu ion a e boiling in 10 % KOH.
A e wa ds, myco hiza ion a ios we e de e mined by scanning 15 cm ine oo s o each
sample o a buscules and esicules unde a mic oscope (400×) using he “magni ied
in e sec ion me hod” (McGonigle e al. 1990).
Soil mic obial ni ogen pool
Soil mic obial ni ogen was ex ac ed om esh soil acco ding o a modi ied
chlo o o m umiga ion–ex ac ion me hod (B ookes e al. 1985). A e chlo o o m umiga ion
(24 h a oom empe a u e), dissol ed o ganic and mic obial N was ex ac ed wi h 50 mL
0.5 M K2SO4 and quan i ied (DIMA TOC-100, Dima ec, Essen, Ge many). Mic obial
biomass and ela i e abundance o mic obial g oups we e measu ed using phospholipid a y
acid (PLFA) analysis as desc ibed (Singh e al. 2006).
Po en ial soil enzyme ac i i ies
Fo soil enzyme ac i i y measu emen s, enzymes in ol ed in ca bon, ni ogen and
phospho us cycling we e selec ed, hus add essing impo an mic obial soil unc ions
(Wald op & Fi es one 2006). The enzyme ac i i ies es ed we e acid phospha ase clea ing
o ganically bound phospha e, cellobiohyd olase, β-xylosidase and β-glucosidase ela ed o he
deg ada ion o plan cell wall componen s and N-ace ylglucosaminidase ep esen ing
chi inases ha deg ade chi in om ungal o a h opod o igin. Soil samples o de e mining
soil enzyme ac i i ies we e collec ed immedia ely a e inishing he d ough manipula ions
(K eyling e al. 2008a). Fou samples pe plo (dep h 0 – 5 cm) we e combined, mixed and
kep a 4 °C un il u he p ocessing wi hin 4 weeks a e sampling. Soil suspensions (0.4 g
esh soil in 40 mL H2O) we e p epa ed om each sample. The assay is based on he
enzyma ic clea age o he below-de ailed me hylumbelli e one (MU) coupled subs a es and
he subsequen de ec ion o MU eleased du ing incuba ion. In b ie , 50 μL pe well o soil
suspensions ( h ee eplica es each sample) we e dispe sed in mic opla es and 100 μL o
subs a e solu ions we e added o s a he eac ions. A e s opping he eac ion wi h 100 μL
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
48
o 2.5 M T is bu e and cen i uga ion, MU concen a ions we e de e mined on a
luo escence spec ome e a exci a ion/emission wa eleng hs o 365/450 nm, espec i ely.
The ollowing enzyme subs a es we e used wi h he incuba ion imes gi en: MUF-phospha e,
20 min; MUF-xyloside, 1 h; MUF-cellobiohyd o u ane, 1 h; MUF-N-ace yl-β-glucosaminide,
40 min; MUF-β-glucoside, 1 h. Subs a e concen a ions in he incuba ion mix we e 500 μM
excep o MUF-cellobiohyd o u ane wi h 400 μM. To accoun o quenching and o calcula e
he amoun o MUF eleased, calib a ion cu es we e included wi h 50 μL o soil samples as
in he incuba ion wells and MUF-solu ions o gi e a inal amoun o 0 - 500 pmol pe well.
Nega i e con ols o au o luo escence o subs a es we e also included. Enzyme ac i i ies a e
exp essed as MUF- elease pe g am soil d y weigh pe hou .
Plan -a ailable soil ni a e and ammonium
Plan -a ailable ni ogen was ex ac ed om ou homogenized, sie ed (< 2 mm),
mixed samples o he uppe soil laye (0-10 cm) o each plo sampled in July using a 1 M KCl
solu ion a e il a ion (Ro h, Ka ls uhe Ge many, Typ 15 A Blauband) (K eyling e al.
2010). Ni a e and ammonium we e quan i ied using low injec ion analysis (FIA, MLE
D esden FIA-LAB).
Lea ca bon o ni ogen a io
Lea ca bon (C), lea ni ogen (N) and C:N a ios we e measu ed om mixed samples
o wo sun-exposed lea es o i e indi idual plan s pe species and plo , sampled in July
(K eyling, Beie kuhnlein & Jen sch 2010). The samples we e o en-d ied o 48 h a 75 °C.
The d y lea es we e ball-milled and subsamples o 1 mg analysed wi h an elemen al analyse
in a mass spec ome e using ConFlo III in e ace. Plan -a ailable ni ogen was ex ac ed
om ou homogenized, sie ed (2 mm) and il e ed (Ro h, Ge many, Typ 15A Blauband)
mixed samples o he uppe soil laye (0–10 cm) o each plo using a 1 M KCl solu ion.
Lea p o ein con en
To al p o ein con en in µg pe mg esh weigh was de e mined as a p oxy o
nu i i e alue o he legume key species H. lana us, which was g owing in all plo s. One lea
sample pe plo was aken on he las day o d ough ea men , ozen in liquid ni ogen and
eeze-d ied o de e mine p o ein-bound amino acids. Amino acids o he p o ein ac ion
we e ex ac ed. Amino acid concen a ions we e measu ed wi h an ion exchange
ch oma og aph (Bio onik, amino acid analyse LC 3000) and p o ein con en was calcula ed
by pooling he con en o each amino acid in he p o ein ac ion.
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
49
Lea ni ogen iso ope signal
Equally aged, sou h- acing lea es o A. ela ius we e collec ed and o en-d ied a 60 °C
o 48 h, and hen ine-milled. Na u al abundance o δ15N and o al ni ogen concen a ion
we e analysed using an elemen al analyse (EA 3000, Eu oVec o , I aly) coupled online o a
ConFlo III in e ace (The mo Elec on, B emen, Ge many) connec ed o an iso ope- a io mass
spec ome e (MAT 253, The mo Elec on, B emen, Ge many) . The δ15N alues we e
calcula ed as: δ15N [‰] = ( Rsample/Rs anda d ) -1)*1000, whe e R ep esen s he a io o
15N:14N iso opes. As s anda d, (ni ogen in) ai was used.
Communi y esponses
In asibili y
In asibili y o he expe imen al communi ies was eco ded h ee imes pe yea : be o e
and a e he d ough manipula ions in ea ly summe , and in all (K eyling e al. 2008c).
In ading plan indi iduals we e collec ed om he inne squa e me e o each plo and
subsequen ly sepa a ed by species. Remo al ook place only a e he i s ue lea es (a e
he co yledons) eme ged, bu mos specimens we e conside ably olde han his and clea ly
es ablished in he s and. A his poin in de elopmen , we expec ed ha numbe o indi iduals
gi e a measu e o es ablished in ade s a he han chance ge mina ions. Fo each plo , he
numbe o indi iduals was de e mined. The plan ed a ge species o he expe imen we e
emo ed om he subsequen analysis. Tes s con i med ha ge mina ion om he soil seed
bank was negligible a e one yea . Thus, in asibili y was only based on species in ading
om he ma ix ege a ion.
Plan composi ional change
The measu emen s o abo e-g ound species-speci ic co e (s. abo e) we e used o
e alua e shi s in he species abundance dis ibu ions o he a i icial plan assemblages.
Composi ional change o each indi idual plo was e alua ed by compa ing he species
abundance dis ibu ion a each ime s ep o he ini ial species abundance dis ibu ion ( i e
weeks a e plan ing) by he B ay–Cu is index.
Compe i i e e ec / acili a i e e ec
The Rela i e Neighbou E ec calcula es he e ec o neighbou s ela i e o he plan
wi h he g ea es pe o mance: RNE = Pcon -Pmix/x wi h x = Pcon i Pcon > Pmix and x = Pmix i
Pmix> Pcon , whe e RNE = Rela i e neighbou e ec (-1 ≤ RNE ≤ +1), Pcon = pe o mance
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
50
pe plan o a plan g owing alone ,Pmix = pe o mance pe plan o a plan g owing in
mix u e. Nega i e alues indica e acili a ion, and posi i e alues indica e compe i ion
(Ma kham & Chanway 1996).
Senescence
Tissue die-back was quan i ied by co e measu emen s o s anding-dead plan o gans
(K eyling e al. 2008d). A pin-poin me hod was applied, eco ding he p esence o plan
o gans in gene al and he p esence o each species sepa a ely a 100 e ically inse ed s eel
needles. These alues we e ea ed as pe cen age co e . The measu emen was epea ed ou
imes o e he cou se o he ege a ion pe iod.
Va iabili y in leng h o lowe ing
Fo each species, weekly obse a ions o he lowe ing s a us o ou indi iduals pe
plo and species we e ca ied ou (Jen sch e al. 2009). Indi iduals we e coun ed as
‘ lowe ing’ when he an he s we e isible in a leas one lowe . Flowe ing leng h was
calcula ed as he di e ence be ween he da es o he 25 and 75 pe cen ile o he lowe ing
cu e o e ime. Va iabili y in leng h o lowe ing was ob ained as he s anda d de ia ion
be ween all species o each ea men (d ough and con ol) sepa a ely. S a is ical
signi icance o di e ence in a iabili y was e alua ed by he Le ene es .
Va iabili y in lowe phenology
Flowe phenology was ob ained om he same da a as leng h o lowe ing (see abo e).
As a su oga e, he mid- lowe ing da e was calcula ed o each species and plo , i.e. he da e
o he 50 pe cen ile o he lowe ing cu e o e ime. Va iabili y in lowe phenology was
exp essed as he s anda d de ia ion be ween all species o each ea men (d ough and
con ol) sepa a ely. S a is ical signi icance o di e ence in a iabili y was e alua ed by he
Le ene es .
Resis ance o he bi o y (phenol con en )
Fo analysis o o al soluble ca bohyd a es and o al phenolics, h ee mixed samples o
a leas wo plan s pe plo we e aken a he end o he d ough pe iod, immedia ely ozen in
liquid ni ogen and lyophilized (n=15). Thi y milig ams we e ex ac ed in 50 % me hanol.
To al soluble ca bohyd a es we e analysed using he an h one me hod wi h glucose as a
s anda d. Ex inc ion was measu ed a 620 nm. To al phenols we e analysed using
FolinCiocal eu’s eagen and ca echin as a s anda d and measu ing ex inc ion a 750 nm.

Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
51
P ima y consume abundance
Richness was sampled in June in one ci cula a ea (40 cm diame e ) in each g assland
plo using a D-Vac suc ion sample (eco ech GmbH, Bonn, Ge many). Fo each plo , he
sampling bag was emo ed and all sampled ma e ial was s o ed in e hanol. A h opod
samples we e quan i ied as he o al numbe o indi iduals and iden i ied a leas o o de
le el. Howe e , some axa we e iden i ied o he amily le el ( amilies wi hin he Coleop e a,
Hemip e a, mos Hymenop e a) and in one case o genus le el (Psylliodes [Ch ysomelidae].
The use o highe axonomic le els has been shown o p oduce a good app oxima ion o o al
species ichness (Biaggini e al. 2007).
S a is ical Analyses
Linea Models combined wi h analysis o a iance (ANOVA) we e applied o es o
signi ican di e ences be ween g oups a single poin s o ime, while aking he spli -plo
design in o accoun . Homogeneous g oups o ac o combina ions (d ough manipula ion,
ege a ion ype, di e si y le el) we e iden i ied by Tukey HSD pos hoc compa isons. Le el
o signi icance was se o p<0.05. S a is ical signi icance o di e ence in a iabili y o leng h
in lowe ing was e alua ed by he Le ene es .
Fo ime se ies, Linea Mixed-E ec s Models we e employed o es o e ec s o
d ough manipula ion and di e si y and hei espec i e in e ac ions while aking he spli -plo
design and he epea ed measu es in o accoun ( ime used as andom ac o ). When no
signi ican in e ac ion was ound, he model was simpli ied by using only he d ough
manipula ions as ixed e ec s and ime as andom e ec . Signi icance o di e ences (p <
0.05) was e alua ed by Ma ko Chain Mon e Ca lo sampling o 1000 pe mu a ions. Linea
Mixed-E ec s Models we e conduc ed wi h he unc ion ‘lme ’ (Ba es & Sa ka 2007).
P io o s a is ical analysis, da a was log- o squa e- oo - ans o med, i condi ions o
no mali y we e no me , o o imp o e homogenei y o a iances. Bo h cha ac e is ics we e
es ed by examining he esiduals e sus i ed plo s and he no mal qq-plo s o he linea
models. All s a is ical analyses we e pe o med using R.
Resul s
The e ec s o d ough on all measu ed ecosys em p ope ies a e summa ized in Fig. 1 using
esponse a ios o s anda dize he e ec size o he se e e d ough ea men .
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
52
Figu e 1: E ec s o ecu en se e e d ough e en s on 32 esponse pa ame e s o ganized in o ecosys em
unc ions. All da a we e collec ed a he EVENT I expe imen al si e (Jen sch, K eyling & Beie kuhnlein
2007) in Cen al Eu ope du ing he yea s 2005 - 2009. A pa ame e is ma ked as signi ican ( illed black
ba ), i da a o a leas one yea showed signi ican di e ences be ween d ough and ambien condi ions
(ANOVA). Da a shown ep esen maximum e ec s om yea s wi h highes d ough e ec s, a e aged o e
all h ee expe imen al g assland communi ies. Fo e e ences o published de ails please e e o Ma e ials
and me hods sec ion
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
53
Wa e egula ion
Se e e d ough signi ican ly educed soil mois u e du ing he manipula ion pe iods in
all yea s (Figs 1, 2).
Figu e 2 Soil mois u e in he EVENT expe imen
a -2 o -7 cm du ing manipula ion (ligh g ey
boxes) and eco e y a e ex eme d ough o
con ol (black line) and d ough (g ey line).
MJJA = May, June, July, Augus . Plan a ailable
wa e is shown be ween he dashed lines:
pe manen wil ing poin (pF = 4.2) and ield
capaci y (pF = 1.8). See Ma e ials and me hods
o echnical de ails.
A high a iabili y bo h wi hin yea s and be ween yea s is e iden due o in e -annual
a iabili y o p ecipi a ion (Table 2). E en hough absolu e minima in soil mois u e we e
simila o d ough and con ol in mos yea s, soil mois u e o he d ough plo s emained
conside ably longe below he app oxima e pe manen wil ing poin (pF = 4.2) o he soil
subs a e. The manipula ion e ec anished wi hin days o all yea s excep 2009, whe e a lag
phase o abou wo mon hs un il Augus occu ed.
Table 2 Tempe a u e and p ecipi a ion sums (added daily amoun ) o each yea un il he s a o he
d ough manipula ion and he espec i e al e a ion om he long- e m mean (1971-2000, da a: Ge man
Wea he Se ice s a ion Bay eu h)
yea empe a u e sum
(1 Janua y o s a
o manipula ion)
ela i e change o
empe a u e sum
compa ed o long-
e m mean (%)
p ecipi a ion sum
(1 Janua y o s a
o manipula ion)
ela i e change o
p ecipi a ion sum
compa ed o long-
e m mean (%)
2005 824.7 -3 259.7 -9
2006 394.7 -38 208.3 +10
2007 978.7 +77 258.6 +9
2008 757.6 +40 282.2 +19
2009 574.9 +4 246.4 +4
Fu he , d ough dec eased lea wa e po en ial, while inc easing lea ca bon iso ope signal in
some species (Figu e 1).
P ima y p oduc ion
A he le el o he g assland communi y o ecosys em, espec i ely, local, annually
ecu en 100-yea and 1000-yea ex eme d ough e en s had no signi ican e ec on a ious
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
54
p ocesses ha con ibu e o p ima y p oduc ion in any o he i e yea s om 2005 o 2009
(Figs 1, 3). Su p isingly, nei he abo e-g ound p ima y p oduc ion (ANPP), no g een co e
o ege a ion o below-g ound p oduc ion eco ded as oo leng h in he main oo ing ho izon
we e a ec ed by d ough (Figs 1, 3).
Figu e 3: (a) Abo e-g ound Ne P ima y P oduc ion
(ANPP) , (b) co e o g een biomass, and (c) oo
leng h o e i e g owing seasons (mean ± SE o e all
species composi ions in g assland, n = 15 pe da a
poin ). An as e isk ma ks signi ican ea men e ec s
(ANOVA, Tukey HSD pos hoc compa ison: p < 0.05)
Fu he , he e was no signi ican d ough e ec on biomass p oduc ion o he ni ogen- ixing
plan L. co nicula us (Figu e 1).
Ca bon ixa ion
D ough inc eased he maximum up ake capaci y (GPPmax) in g assland by 36 %
(Figu e 1). The soil espi a ion a e (Reco calcula ed by he model was lowe unde d ough
han unde ambien condi ions. Soil espi a ion was sligh ly bu no signi ican ly dec eased a
he end o he d ough .
2005 2006 2007 2008 2009
0
100
200
300
400
Yea
ANPP [g m-² a-1]
0
20
40
60
80
100
G een co e [%]
Ambien
D ough
Roo leng h [cm 4 cm-2]
n.s.
n.s.
(a)
(b)
0
1
2
3
4
5
6(c)
n.s.
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
61
Conclusion
Ou expe imen al da a demons a e ha wea he ex emes ini ia e ecosys em-
egula ing unc ions such as wa e and nu ien cycling, gas exchange and composi ional
dynamics while main aining p ima y p oduc ion. They indica e an impo an con ibu ion o
ecological complexi y o he main enance o p oduc i i y in he ace o inc eased empo al
clima e a iabili y and ex ao dina y wea he e en s. Howe e , single species eac ions can
no be ansla ed di ec ly o he communi y and ecosys em le el. A po en ial eason o
di e en d ough impac s on a ious ecosys em p ope ies may lie in he empo al hie a chy
o as e sus slow esponse pa e ns. In ou empe a e g assland, we obse ed he ollowing
esponse dynamics wi hin hal a decade o ecu en d ough e en s: e y as al e a ion o
soil mois u e s a us, subsequen as change in nu ien cycling and gas exchange, slow
species-speci ic esponse in p ima y p oduc ion, ine ia in communi y p oduc i i y.
Such da a on mul iple esponse pa ame e s wi hin clima e change expe imen s os e
he unde s anding o mechanisms o esilience, o syne gisms o decoupling in
biogeochemical p ocesses, and o undamen al esponse dynamics o d ough a he ecosys em
le el.
As i was he case wi h he open ques ions on he consequences o he c isis o biodi e si y,
we see his complexi y in s udying impac s o clima e ex emes as a new chance o a boos in
ecological heo y. Addi ionally, comp ehensi e s udies on he complex esponses will help
de eloping coping s a egies o he adap ed managemen o hese ecosys ems.
Fu u e challenges consis o analysing esponses o mul iple ecosys em unc ions and
a mul iple le els o o ganiza ion wi h he goal o assessing how hey in e ac o in luence
eme gen ecosys em p ope ies, such as ecosys em unc ion and s abili y. The obse ed
s abili y in p ima y p oduc ion in he ace o ecu en se e e d ough does no mean ha he
esponses a he ecosys em le el a e null. On he con a y, he obse ed changes in ecosys em
egula ing unc ions in e ms o gas exchange, nu ien cycling, wa e egula ion and
communi y s abili y sugges a p ominen ole o ex eme wea he e en s in ecosys em
esponse o clima e change. Howe e , modelling he beha iou o ecosys ems du ing and a e
ex eme clima ic e en s a la ge spa ial scales and o e longe pe iods o ime equi es mo e
in-dep h knowledge on possible esponse mechanisms a he le el o plan communi ies.
Po en ial epigene ic, physiological o ophic esponses need o be igo ously u he explo ed
expe imen ally. Labo a o y s udies on molecula mechanisms ha e o be ela ed o s udies

Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
62
wi h he same species in he ield. Field s udies mus in eg a e a ious le els o unc ional
di e si y (Beie kuhnlein e al. in p ess). Pheno ypical di e si y o popula ions has o be
conside ed. Li e cycles o plan species and coho s can be o c ucial impo ance. G adien s in
soil ypes ha e o be in eg a ed. Then, we can each a be e unde s anding o he mechanisms
ha a e ini ia ed in plan communi ies by ex eme e en s.
Fu u e wo k is needed o elucida e he ole o biodi e si y and o bio ic in e ac ions in
modula ing ecosys em esponse o ex eme wea he e en s. Fu he , we need mo e da a on
impac s o clima e ex emes on mul iple ecosys em p ope ies om a ious ecosys ems and
biomes, in o de o os e he sea ch o gene ali y ac oss di e en ca ego ies o esponse.
He e, a majo challenge is o assess he speed o esponse ac oss a ious pa ame e s,
including long- e m eedbacks, i.e. caused by a ni ogen-dependen eedback on p oduc i i y
(Haddad e al. 2002).
Gene ally, scien is s a e challenged by ela ing he ecosys em p ope ies measu ed
(he e: ne ecosys em exchange, biomass abo e and below g ound, ca bon ixa ion by
pho osyn hesis, nu ien a ios) o ecosys em unc ions and se ices, such as p oduc i i y,
ca bon ixa ion, nu ien cycling, decomposi ion and wa e egula ion. Measu ing ecosys em
se ices is a as -de eloping esea ch a ea wi h many deba es on how o assess he se ices
adequa ely.
Acknowledgemen s: The con ibu ion o a ious wo king g oups o he measu emen s in he
EVENT expe imen gi es us a unique oppo uni y o b ing bi s and pieces oge he . We hank
J. Bö che -T eschkow, M. Ewald, N. He old, Z. Hussein Y. Li, M. Mede e , C. Mülle , L.
Muelle , S. Neugebaue , D. P ab, K. Simmnache , H. Skiba, S. Wal he , M. Wenigmann, D.
Wul and many s uden helpe s o assis ance wi h da a mining in he ield and ui ul
discussions.
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Online Suppo ing In o ma ion
Table S1: Sea ch i ems o sea ching he ISI Web o Science® Da abase o publica ions on wea he
e en s and clima e ex emes. As e isks a e place holde s wi hin he sea ch s ing
Main ca ego y Sea ch i ems in ISI Web o Knowledge
F os * os e en * OR se e e nigh os * OR *sp ing os * OR eeze- haw* OR
haw- eeze* OR la e os * OR se e e os * OR *g ound os * OR ex eme
os * OR ex eme cold*
Hea & d ough hea wa e* OR hea wa e* OR se e e hea * OR * empe a u e e en * OR d y
spell* OR ex eme hea * OR *win e wa ming* OR wa m* win e OR summe
d ough * OR sp ing d ough * OR au umn d ough * OR se e e d ough *
S o m ex eme s o m* OR *win e s o m* OR hu icane* OR yphoon* OR cyclone*
OR o nado* OR s o m su ge* OR *winds o m* OR *wind s o m* OR * opical
s o m* OR ice s o m*
Hea y ain ex eme lood* OR summe lood* OR ex eme ain* OR o en ial ain* OR
ex eme p ecipi a ion OR * ain all e en * OR hea y ain* OR hail* OR we
spell*
Ex eme ex eme e en * OR ex eme wea he e en * OR clima * ex eme* OR
ex eme me eo ological e en * OR ex eme wea he * OR ex eme clima *
e en *
Table S2: Links o sea ching he ISI Web o Science® Da abase o publica ions on wea he e en s and
clima e ex emes
Links Sea ch i ems in ISI Web o Knowledge
Main i ems
in “Topic”
F os , Hea & d ough , S o m, Hea y ain, Ex eme
AND
in “Topic”
we land* OR loodplain* OR pea * OR bog* OR en* OR swamp* OR mi e OR
g assland* OR meadow* OR pas u e* OR hea h* OR sh ubland* OR o es * OR
woodland* OR und a OR aiga OR sa anna* OR ma sh* OR s eppe OR dese *
OR aqua ic* OR limn*
*bi d* OR a ian OR insec * OR bu e ly* OR bee le* OR a h opod* OR mo h*
OR amphibian* OR ep ile* OR mollusc* OR mollusk* OR e eb a e* OR
*in e eb a e* OR mammal*
AND
in “Topic”
clima * change* OR global change* OR clima * wa ming
NOT
in “Topic”
palaeo* OR paleo* OR pleis ocene OR holocene

Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
67
Table S3: Sampling yea s o all esponse pa ame e s p esen ed in Figu e 1. Gi en a e da a om yea s
wi h maximum d ough e ec
Ecosys em p ope y Yea o sampling wi h
maximum d ough e ec
Ecosys em se ice
Abo e-g ound p oduc ion (ANPP) 2005
Ni ogen ixing plan s 2009
Plan co e 2009
P ima y p oduc ion
Below-g ound biomass 2007
shoo / oo - a io 2006
Gas exchange Maximum ca bon up ake capaci y 2005
Pho osyn he ic pe o mance 2008
Lea gas exchange 2007
Soil espi a ion 2010
Nu ien cycling Decomposi ion a e 2007
Myco hiza ion a e 2008
Soil mic obial biomass 2008
Soil enzyme ac i i y 2006
Plan a ailable NO3- 2008
Plan a ailable soil NH4 2008
soil mic obial N 2009
Lea C/N- a io 2009
Lea p o ein con en 2009
Lea ca bohyd a e con en 2009
Lea ni ogen iso ope signal 2007
1° consume abundance 2008
Soil mois u e 2009
Wa e egula ion
Lea wa e po en ial 2008
Lea ca bon iso ope signal 2007
Communi y esponses In asibili y 2006
Plan composi ional change 2005
Senescence 2006
Va iabili y in leng h o lowe ing 2006
Va iabili y in lowe phenology 2008
Resis ance o he bi o y 2009
Compe i i e e ec 2007
Facili a i e e ec 2007
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
68
(a)
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
69
(b)
(c)
Manusc ip 1: Clima e ex emes ini ia e ecosys em egula ing unc ions while main aining p oduc i i y
70
(d)
Figu e S1: Resea ch on ecological e ec s o clima e ex emes and wea he e en s based on publica ions
ound in he ISI Web o Science ( o sea ch de ails see Table 2) (a) Tempo al de elopmen o he numbe
o publica ions on clima e ex emes (n=380) in he las wo decade (shown is only he las decade); o al
yield 1134 pee - e iewed pape s (b) S udied ex eme wea he e en s (n=464 inlcuding double o iple
assignmen s) o he ele an pee - e iewed pape s (n=380) yielded by he li e a u e s udy. 24 publica ions
did no speci y he e en . (c) Resea ch ac i i y in he h ee main biomes by p opo ion o publica ions
based on 380 pee - e iewed pape s pa icula ly s udying e ec s o clima e ex emes on ecosys em
unc ions. G assland includes dese s, pea and we lands. Sh ubland includes und a. Any one pape may
ha e been assigned o mul iple subjec a eas. (d) S udied e ec s o ex eme wea he e en s on ecosys em
p ope ies a anged by ecosys em se ices and unc ions based on 380 pee - e iewed pape s pa icula ly
s udying e ec s o clima e ex emes on ecosys em unc ions
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
77
and he le els we e shi ed e e y second day, pu ing he lowes le el o he highes place and
all o he le els one le el lowe . The plan lea es om one plo we e ed o he la ae in ou
pe i dishes (8 la ae in o al). Ca e pilla s we e able o ea ad libi um, as special ca e was
aken ha he g ass inside one pe i dish was ne e o ally consumed. Lea es we e eplaced a
leas e e y second day. We eco ded he mo ali y o he la ae, he de elopmen ime un il
pupa ion, he weigh o he pupae one day a e pupa ion and pupal mo ali y. Th ough he
isola ion in clima e chambe s we we e able o a ibu e he esponses in he bi o e
pe o mance o di e ences in plan compounds, as opposed o when eeding is conduc ed
unde ield condi ions and i is impossible o disen angle he e ec s o plan nu ien s,
compensa o y eeding and di ec wea he e ec s on he bi o es (Go e de e al. 2002).
S a is ical analysis
Chemical lea ai s we e analyzed using a wo-way ANOVA wi h wea he ea men
and communi y composi ion as ixed ac o s. Addi ionally, we included he numbe o
columns and ows as andom ac o s. This au oma ically implemen s he nes ing o
composi ion wi hin ea men -blocks in he mixed e ec model (Fa away 2006; Do mann and
Kühn 2008). I se e al samples pe plo we e aken, as was he case wi h phenolics,
ca bohyd a es and condensed annins, hen he plo numbe was addi ionally included as a
andom ac o in he mixed model, o a oid any pseudo- eplica ion.
De elopmen al ime and pupal weigh we e analyzed using linea mixed e ec models
wi h he pe i dish nes ed wi hin he plo nes ed wi hin he ea men block as a andom ac o ,
in addi ion o he ow and he column o he ea men blocks as andom ac o s. La al and
pupal mo ali y we e analyzed using gene alized mixed e ec models wi h binomial
dis ibu ion and o he wise he same model o mula as o o he de elopmen al ai s.
The signi icance le els in he mixed e ec models we e e alua ed by Ma ko Chain
Mon e Ca lo sampling o 1000 pe mu a ions (Baayen 2009; package language R).The
signi icance o he ixed ac o s o he gene alized mixed e ec models was de e mined by
compa ing he null model, wi hou any ac o s, o he simples ac o ial model, in which non-
signi ican e ms had been emo ed by backwa ds s epwise selec ion. P io o all analyses,
da a we e ans o med acco dingly, i he assump ions o ANOVA, homogenei y o a iances
and no mali y, we e no me (C/N a io and ni ogen con en : log- ans o med; RWC: a csin-
squa e oo - ans o med).
To de e mine he ela ionship be ween chemical lea ai s and de elopmen ai s, we
applied hie a chical pa i ioning, as lea chemical ai s a e o en collinea (Schädle e al.

Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
78
2003). In hie a chical pa i ioning, he independen in luence and he join in luence ( he
in luence om being co ela ed o ano he explana o y a iable) o explana o y a iables is
calcula ed by compa ing he model- i s o models wi h and wi hou he pa icula a iable
(Mac Nally 2002; Do mann and Kühn 2008). To de e mine co ela ions be ween he di e en
de elopmen ai s, a co ela ion analysis was used o co ela e pupal weigh wi h
de elopmen al ime, and logis ic eg ession was used o in es iga e he ela ionship be ween
pupal mo ali y and pupal weigh .
All s a is ical analyses we e pe o med using R 2.11.0 (R De elopmen Co e Team
2010). Fo mixed e ec models we used he so wa e package lme4 (Ba es & Mechle 2010),
and o mul iple pos -hoc compa isons he package mul comp was used (Ho ho n e al. 2008).
Resul s
Soil mois u e
The ege a ion pe iod o he yea 2009 (Ap il 1s – Oc obe 31s ) wi h a o al sum o
459 mm o p ecipi a ion was sligh ly we e han he long- e m a e age p ecipi a ion sum o
437 mm o he ime pe iod 1971-2000 (Da a: Ge man Wea he Se ice). Soil mois u e ell
mo e quickly du ing he i s hal o he d ough pe iod compa ed o he second hal , bu ose
quickly again a e he d ough pe iod was o e (Fig.1).
Day o he yea
140 150 160 170 180
0,00
0,05
0,10
0,15
0,20
0,25
0,30 D ough
Con ol
Soil mois u e [ ol % ]
0
5
10
15
20
25
A e age Tempe a u e [°C ]
Day o he yea
140 150 160 170 180
0,00
0,05
0,10
0,15
0,20
0,25
0,30 D ough
Con ol
D ough
Con ol
Soil mois u e [ ol % ]
0
5
10
15
20
25
A e age Tempe a u e [°C ]
Fig. 1 Cou se o soil mois u e in d ough -exposed plo s (black ci cles) and con ol plo s (da k-g ey
squa es), and a e age daily empe a u es, assessed a a heigh o 1, 20 m (ligh g ey ba s). Da a a e shown
om he i s day o he d ough manipula ion (day o he yea 140=May 20 h, 2009) un il wo days a e
he ex eme d ough ended, indica ed by he black e ical line (day o he yea 182=July 1s , 2009). The
a e age o hou ly eadings om i e senso s pe ea men we e aken he e (n=5)
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
79
The e ec o d ough and communi y composi ion on chemical lea ai s
D ough signi ican ly dec eased RWC by 8 % (Fig. 2a), p o ein con en by 23 % (Fig.
2b), ni ogen concen a ions by 26 % (Table 3) and phenols by 7 %, when compa ed o he
con ol ea men (Fig. 2c) (see Table 2 o s a is ical de ails). Fu he mo e, d ough
signi ican ly inc eased he C/N a io by 24 % and he soluble ca bohyd a es by 32 % (Fig. 2d,
e). Condensed annins howe e we e no al e ed by d ough manipula ion (Table 2).
CD
RWC [%]
020
40 60 80 100
(a)
CD
P o ein con en [µg/mg FW]
012345
(b)
CD
Phenols [nmol/mg]
0 50 100 150 200
(c)
CD
C/N a io [%]
0 10203040
(d)
CD
Ca bohyd a es [nmol/mg]
0 100 200 300
(e)
Wea he ea men
CD
RWC [%]
020
40 60 80 100
(a)
CD
P o ein con en [µg/mg FW]
012345
(b)
CD
Phenols [nmol/mg]
0 50 100 150 200
(c)
CD
C/N a io [%]
0 10203040
(d)
CD
Ca bohyd a es [nmol/mg]
0 100 200 300
(e)
Wea he ea men
CD
RWC [%]
020
40 60 80 100
(a)
CD
P o ein con en [µg/mg FW]
012345
(b)
CD
Phenols [nmol/mg]
0 50 100 150 200
(c)
CD
C/N a io [%]
0 10203040
(d)
CD
Ca bohyd a es [nmol/mg]
0 100 200 300
(e)
CD
RWC [%]
020
40 60 80 100
CD
RWC [%]
020
40 60 80 100
(a)
CD
P o ein con en [µg/mg FW]
012345
CD
P o ein con en [µg/mg FW]
012345
(b)
CD
Phenols [nmol/mg]
0 50 100 150 200
(c)
CD
C/N a io [%]
0 10203040
CD
C/N a io [%]
0 10203040
(d)
CD
Ca bohyd a es [nmol/mg]
0 100 200 300
CD
Ca bohyd a es [nmol/mg]
0 100 200 300
(e)
Wea he ea men
Fig. 2 Di e ences in a) RWC (n=5), b) he p o ein con en (n=5), c) he o al soluble phenol con en (n=15),
d) he C/N a io (n=5) and e) he o al soluble ca bohyd a es (n=15) in lea es o Holcus lana us unde
d ough (ligh g ey ba s, D) compa ed o con ol (whi e ba s, C). As e isks indica e he le el o
signi icance: * p<0.05; **p<0.01; ***p<0.001. Means ± 1 SE a e shown
Communi y composi ion signi ican ly a ec ed he con en o condensed annins and
phenols (Table 2) and ma ginally signi ican ly a ec ed he con en o soluble ca bohyd a es
(p=0.07) and p o eins (p=0.01).
Table 2 ANOVA esul s o he e ec s o d ough ea men , communi y composi ion and, i signi ican ,
hei in e ac ion on RWC, o al soluble p o ein con en , ni ogen concen a ions, C/N a io (n=5,
espec i ely), o al soluble ca bohyd a es, o al soluble phenols and condensed annins (n=15,
espec i ely). Values o in e ac ion a e only shown, when signi ican , as o he wise, he in e ac ion e m
was excluded om he mixed model (d =deg ees o eedom, MS=mean sum o squa es). As e isks
indica e le el o signi icance: `p<0.01; * p<0.05; **p<0.01; ***p<0.001
RWC p o ein ni ogen C/N ca bohyd a es phenols cond. annins
d MS F d MS F d MS F d MS F d MS F d MS F d MS F
d ough 1 0.18 28.4*** 1 7.51 7.4* 1 1.02 65.1*** 1 1.13 76.6*** 1 106533 68.3*** 1 5871 11.3** 1 76.4 0.6
composi ion 3 0.01 1.4 3 2.29 2.26` 3 0.01 0.7 3 0.01 0.8 3 3755 2.4` 3 2547 4.9** 3 516.6 4.2**
in e ac ion - - - - - - 3 0.06 4.1* 3 0.04 2.6` - - - - - - - - -
esiduals 30 0.01 32 1.01 32 0.02 32 0.01 97 1558 97 518 97 124.5
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
80
The condensed annins in H. lana us om 4+ communi ies we e signi ican ly lowe
han he condensed annins in lea es om 2- and 4- communi ies (Fig. 3a). Phenols we e
educed in H. lana us g owing in legume communi ies (4+), when compa ed o H. lana us
om monocul u es (1-) (Fig. 3b). Ca bohyd a es we e ma ginally signi ican ly inc eased in
legume communi ies (4+) when compa ed o wo-species communi ies wi hou legume (2-)
(Fig. 3c), while p o ein con en was ma ginally signi ican ly lowe in ou -species
communi ies wi hou legume (4-) when compa ed o legume communi ies (4+)(Fig. 3d).
Fig. 3 E ec s o plan communi y composi ion
on a) he con en o condensed annins (n=15),
b) he o al soluble phenol con en (n=15), c) he
o al soluble ca bohyd a es (n=15) and d) he
o al soluble p o eins (n=5) in lea es o Holcus
lana us (1-: monocul u e, 2-: wo g asses, 4-: 2
g asses, 2 he bs; 4+: wo g asses, 1 he b, 1
legume). Di e en le e s indica e signi ican
di e ences be ween he communi ies (p<0.05).
E ec s o ca bohyd a e and p o ein con en
we e only ma ginally signi ican (p<0.1)
As a as lea ni ogen concen a ion was conce ned, communi y composi ion
signi ican ly in e ac ed wi h he d ough ea men , as ni ogen concen a ion was educed in
all communi ies excep o he 4+ communi ies in esponse o d ough (Table 3).
Table 3 In e ac i e e ec s o communi y composi ion and he d ough ea men on he ni ogen
concen a ion in lea es o H. lana us (n=5). Means ± 1 s anda d de ia ion a e gi en. As e isks behind he
communi y label indica e a signi ican d ough e ec in he espec i e communi y (p<0.05)
composi ion
1- * 2- * 4- * 4+
con ol 1.99 ± 0.31 1.90 ± 0.31 1.88 ± 0.31 1.69 ± 0.30
d ough 1.26 ± 0.32 1.45 ± 0.32 1.26 ± 0.32 1.51 ± 0.31
Cond. Tannins [nmol/mg]
010 20 30 40
1- 4-
2- 4+
Phenols [nmol/mg]
0 50 100 150 200
1- 4-
2- 4+
Ca bohyd a es [nmol/mg]
0100 200 300
P o ein con en [µg/mg FW]
012345
(a) (b)
(d)(c)
Communi y composi ion
ab
aa
b
aab b
ab
Cond. Tannins [nmol/mg]
010 20 30 40
1- 4-
2- 4+
Phenols [nmol/mg]
0 50 100 150 200
Phenols [nmol/mg]
0 50 100 150 200
1- 4-
2- 4+
Ca bohyd a es [nmol/mg]
0100 200 300
1- 4-
2- 4+
1- 4-
2- 4+
Ca bohyd a es [nmol/mg]
0100 200 300
P o ein con en [µg/mg FW]
012345
(a) (b)
(d)(c)
Communi y composi ion
ab
aa
b
aab b
ab
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
81
De elopmen ai s o S .li o alis and hei ela ion o lea chemical ai s
The communi y composi ion o he a ge g ass was ound o ha e a highly signi ican e ec
on la al mo ali y, wi h la ae eeding om 4- plo s showing a signi ican ly highe mo ali y
han in all o he communi ies (Fig. 4). (P=0.007; Chisq= 12.2). The d ough ea men did no
a ec la al mo ali y.
Fig. 4 E ec o he plan communi y composi ion, in which H. lana us
g ows, on he mo ali y o he 320 S. li o alis la ae. Di e en le e s
indica e signi ican di e ences be ween he communi ies (p<0.05)
(n=20/ ea men combina ion)
The de elopmen ime un il pupa ion was highly signi ican ly inc eased in hose la ae
ha we e ea ed on lea es subjec ed o d ough (p=0.0003; F= 14.7; d =1). Fu he mo e, he
d ough ea men was ound o signi ican ly in e ac wi h communi y composi ion ega ding
de elopmen ime un il pupa ion (p=0.015; F= 3.8; d =3), as he la ae eeding on H. lana us
om he 2- communi ies eac ed wi h he g ea es inc ease du ing he la al s age unde
d ough (Table 4).
Table 4 Di e ences in he du a ion o la al s age o 320 S. li o alis la ae eeding on H. lana us lea es
unde he d ough (ligh g ey ba s, D) and he con ol (whi e ba s, C) ea men s om ou di e en
communi y composi ions. D ough signi ican ly inc eased de elopmen al ime wi h s onges e ec s in 2-
communi ies (n=20/ ea men combina ion). Means ± 1 s anda d de ia ion a e gi en
composi ion
1- 2- 4- 4+
con ol 25 ± 2.5 22 ± 2.4 25 ± 2.3 24 ± 2.3
d ough 25 ± 2.3 27 ± 2.4 27 ± 2.5 26 ± 2.4
The la ae eeding on d ough plan s we e also ound o ha e signi ican ly hea ie
pupae (p=0.033; F= 4.7; d =1) (Fig. 5a) and e ealed a signi ican ly lowe pupal mo ali y
(p=0.007; Chisq= 7.2) (Fig 5b), i espec i e o communi y composi ion.
Mo ali y [%]
02040
60 80 100
Communi y
composi ion
1- 4-
2- 4+
aa
b
a
Mo ali y [%]
02040
60 80 100
Mo ali y [%]
02040
60 80 100
Communi y
composi ion
1- 4-
2- 4+
1- 4-
2- 4+
aa
b
a
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
82
Fig. 5 Di e ences in (a) he pupal weigh and (b) he pupal
mo ali y o S. li o alis la ae ed wi h H. lana us lea es ou o
he d ough (ligh g ey ba s, D) and he con ol (whi e ba s, C)
ea men s. As e isks indica e he le el o signi icance: *P 0.05;
**P 0.01 (n = 20/ ea men combina ion)
Hie a chical pa i ioning showed ha p o ein con en had he g ea es posi i e
in luence on he su i al o la ae (Table 5).
Table 5 Hie a chical pa i ions o he e ec s o lea chemical ai s on de elopmen du a ion, pupal
weigh , la al mo ali y and pupal mo ali y o 320 la ae. The o al explained a iance (R2), he
indi idual e ec on he explained a iance, and he join e ec on explained a iance a e gi en. The la e
quan i ies he e ec ha can be explained by he co ela ion o a speci ic independen a iable wi h o he
independen a iables. + o – behind he mos impo an pa i ions o one pa ame e indica e whe he
he pa ame e s we e nega i ely o posi i ely co ela ed o he lea chemical. RWC=Rela i e wa e
con en ; C/N=C/N a io; ni ogen=ni ogen concen a ion; ca bon=ca bon concen a ion; p o ein= o al
soluble p o eins ; phenols= o al soluble phenols ; annins= condensed annins ; ca bos= o al soluble
ca bohyd a es
RWC C/N ni ogen ca bon p o ein phenols annins ca bos
de elopmen o al 0.143 0.259+ 0.210- 0.014 0.001 0.036 0.029 0.078
ime independen 0.113 0.187 0.118 0.019 0.085 0.013 0.014 0.03
join 0.03 0.071 0.091 -0.005 -0.084 0.024 0.015 0.048
pupal weigh o al 0.229- 0.024 0.04 0.031 0.079 0.094 0.077 0.04
independen 0.178 0.025 0.025 0.035 0.068 0.047 0.048 0.02
join 0.051 -0.001 0.015 -0.003 0.012 0.047 0.029 0.021
la al mo ali y o al 0.01 0.003 0.001 0.001 0.047- 0.006 0.005 0.009
independen 0.009 0.009 0.005 0.004 0.065 0.007 0.01 0.007
join 0.001 -0.006 -0.004 -0.002 -0.018 -0.001 -0.005 0.002
pupal mo ali y o al 0.031 0.097- 0.086 0.017 0 0.049 0.096- 0.042
independen 0.014 0.034 0.028 0.013 0.01 0.023 0.072 0.015
join 0.017 0.063 0.058 0.004 -0.009 0.026 0.023 0.027
The de elopmen ime was mainly posi i ely in luenced by he C/N- a io, and hus
nega i ely by he ni ogen con en , indica ing ha he de elopmen ook longe , he less
ni ogen was in he lea es (Table 5). Pupal weigh was ound o be nega i ely a ec ed by
RWC. Pupal su i al, and hus adul eclosion was posi i ely a ec ed by con en o condensed
annins and by C/N a io. Pupal weigh and de elopmen ime we e no co ela ed (Pea sons
Pupal weigh [g]
00.02 0.06 0.10 0.14
CD
Wea he ea men
Pupal mo ali y [%}
020
40 60 80
CD
(a) (b)
Pupal weigh [g]
00.02 0.06 0.10 0.14
CDCD
Wea he ea men
Pupal mo ali y [%}
020
40 60 80
CDCD
(a) (b)

Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
83
co ela ion coe icien : -0.019; p=0.88). Pupal su i al was posi i ely ela ed wi h pupal
weigh (p=0.023; logis ic eg ession).
Discussion
The ex eme 42-day d ough did no only a ec he wa e con en o he a ge g ass
species, bu also esul ed in changes o almos all o he lea chemical ai s ha we assessed.
These changes o he lea chemicals also clea ly a ec ed he de elopmen ai s o he
he bi o e ca e pilla . Howe e , in con as o ou expec a ions, d ough did no inc ease
de ensi e compounds in he g ass. Fu he mo e, he d ough did no esul in a wo se o e all
he bi o e pe o mance, as adul eclosion and pupal weigh we e e en highe o la ae ed
om d ough ea ed plan s. The communi y composi ion o he a ge g ass also a ec ed
some aspec s o he lea chemical composi ion, bu changes canno clea ly be linked o
inc eased compe i ion o highe s abili y in mo e di e se communi ies. Di e ences mainly
occu ed in legume communi ies: H. lana us g owing in legume communi ies showed no
e ec s o d ough on he lea ni ogen concen a ion and had he lowes con en o condensed
annins and phenols. The communi y composi ion o he a ge g ass a ec ed he mo ali y o
he he bi o e, wi h highes mo ali y in la ae eeding on he g ass g owing in ou -species
communi ies wi hou legume, in which also a end owa ds lowe p o ein con en was
appa en (4-).
Soil mois u e and plan s ess
The ex eme d ough condi ions we e accompanied by a ma ked dec ease in soil
mois u e o e he d ough pe iod, and his in u n clea ly caused plan s ess. In he yea 2009
we did no di ec ly quan i y plan s ess le els, e.g. by de e mining chlo ophyll con en o
maximum quan um yield. Howe e , a educ ion in lea ela i e wa e con en in ou a ge
g ass in all communi ies, along wi h ma ked changes in lea chemicals, indica e acclima ion
p ocesses and s ess eac ions, showing ha he plan s expe ienced s ess (Sinclai and
Ludlow 1985; Cha es e al. 2002).
E ec o d ough and communi y composi ion on chemical lea ai s
A dec ease in p o eins and ni ogen, along wi h an inc ease in he C/N a io unde
se e e d ough condi ions has also been obse ed in o he s udies in es iga ing d ough
e ec s on lea chemicals (Shu e e al. 1998; Liu e al. 2008). Howe e , his is no in
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
84
acco dance wi h he so-called “plan s ess hypo hesis”, which assumes a ailable ni ogen o
inc ease unde plan s ess (Whi e 1984). Ne e heless, as ni ogen up ake is linked o wa e
up ake, a dec ease in ni ogen up ake, and he e o e also p o ein con en unde d ough , is no
su p ising. An inc ease in ca bohyd a es unde d ough could be a ibu ed o osmo ic
adjus men in he cou se o d ough acclima ion. Soluble ca bohyd a es om s a ch
deg ada ion ac as compa ible solu es o p e en u go loss in plan cells (Cha es e al. 2002;
Regie e al. 2009). The dec ease in o al phenolics unde d ough does no suppo he idea
ha plan s unde s ess use he su plus om ca bohyd a es (due o es ic ed g ow h while
pho osyn hesis is s ill assimila ing ca bon) o accumula e mo e C-based de ence compounds
(He ms and Ma son 1992). Howe e , a educ ion o phenols in esponse o d ough has also
been epo ed by Shu e e al. (1998). In ou s udy, he need o osmo ic adjus men unde
ex eme d ough migh ha e been a eason no o accumula e phenols, bu a he in es he
su plus o ca bon-based compounds in soluble ca bohyd a es.
As communi y composi ion can al e esou ce pa i ioning be ween plan s, i migh
also change he o age quali y o lea issue. Bo h phenols and condensed annins we e ound
o be lowes in he communi ies ha included one legume species. This kind o educ ion in
de ence compounds can be explained by he po en ially highe ni ogen a ailabili y in legume
communi ies caused by N2- ixing in oo -nodules, enabling highe g ow h a es a he expense
o lowe de ence by C-based compounds (He ms and Ma son 1992). The end owa ds
highe p o ein con en in lea es om legume communi ies compa ed o ou -species
communi ies wi hou legumes suppo he idea o an inc ease in ni ogen a ailabili y in
legume communi ies and inc eased compe i ion o ni ogen in communi ies con aining ou
species, bu no legume. Mo eo e , labelling s udies indica e a di ec up ake o legume-de i ed
ni ogen by g asses (Gubsch e al 2011). O e all, he ni ogen concen a ion o g asses
g owing in legume communi ies (4+) was no a ec ed by d ough , bu showed changes in
esponse o d ough in he o he communi ies. The highe s abili y in his communi ies and he
possible e iliza ion e ec o he legume suppo o he s udies which ha e shown an
enhancemen o he ni ogen a ailabili y o plan s g owing in legume communi ies (Spehn e
al. 2002; Tempe on e al. 2007; Dybzinski e al. 2008).
De elopmen al ai s o S. li o alis and hei ela ion o lea chemical ai s
The mo ali y o la ae was high, as he expe imen was s a ed immedia ely a e
ha ching o he la ae, when hey a e qui e ulne able. Fu he mo e, H. lana us seemed o be
a sub-op imal ood sou ce o S. li o alis, as la ae he same age om he same egg s ain
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
85
g ew be e when eeding on Plan ago lanzeola a and T i olium p a ense, unde o he wise
simila condi ions. Ne e heless, as we expec ed mo ali y o be high, due o he esul s om a
p io ial expe imen , we included enough eplica es in o de o ob ain a subs an ial da a se
o s a is ical analysis.
The epo ed changes in lea me aboli es, ela ed o he d ough ea men and
di e ences in he communi y composi ions, clea ly had an e ec on he bi o e pe o mance.
Mo ali y o la ae was highe in ou -species communi ies wi hou a legume compa ed o he
o he communi ies. Hie a chical pa i ioning showed ha he mo ali y a e depended mos ly
on he p o ein con en . These da a hin owa ds a cen al ole o p o eins o he su i al o he
ea ly ins a s. O he s udies, oo, showed poo la al su i al unde low ni ogen
concen a ions (Mye s and Pos , 1981; Ca es 1987; de B uyn e al. 2002).
La al de elopmen up o pupa ion was signi ican ly longe o la ae ed on d ough -
s essed plan s, which migh inc ease p eda ion isk and hus mo ali y unde na u al
condi ions (Ben ey and Denno 1997). In acco dance wi h Fische and Fiedle (2000) and wi h
Mo ehouse and Ru owski (2010), he p olonged de elopmen al ime o he la ae was linked
o a educed N-a ailabili y in d ough -s essed lea es. I migh be ha la ae ed longe on N-
limi ed g ass o each a ce ain “g ow h a ge ” (Raubenheime and Simpson 1997). Such
compensa o y eeding on low quali y issue may be a common phenomenon (Schädle e al.
2007b).
D ough also had posi i e e ec s on he bi o e pe o mance, i espec i e o
communi y composi ion: The inc eased pupal weigh migh be explained by a highe up ake
o ene gy, as ca bohyd a e con en s in lea es inc eased unde d ough and as la ae ed longe
on d ough plan s, possibly caused by lowe ed ni ogen con en s. Thus, he “imbalanced die ”
when eeding on d ough s essed plan s caused i s ly compensa o y longe eeding, o each
a ce ain ni ogen le el necessa y o de elopmen , and secondly led o a highe ene gy
up ake and highe pupal weigh s (Raubenheime and Simpson 1997). RWC was nega i ely
ela ed o pupal weigh , as p esumably wa e dilu es nu ien s o ca bohyd a es in well-
wa e ed plan s. The inc eased pupal weigh was co ela ed wi h a lowe pupal mo ali y o he
la ae ha had been ed om d ough -s essed plan s, which is in acco dance o o he s udies
(Fische and Fiedle 2000).
I should be ei e a ed ha he samples o he lea chemical analysis we e aken on he
las wo days o he d ough ea men , whe eas he la al de elopmen las ed om
app oxima ely en days be o e o en days a e he d ough . Thus, la ae ed on lea es ha
expe ienced milde s ess le els in hei la e s ages, which migh ha e alle ia ed he e ec s o
Manusc ip 2: How do ex eme d ough and plan communi y composi ion a ec hos plan
me aboli es and he bi o e pe o mance
86
ex eme d ough on lea chemical ai s (Hube y and Denno 2004). Fu he s udies wi h a
highe empo al esolu ion would make i possible o in es iga e he cou se o changes in lea
ai s o e he whole ange o plan s ess esponses.
Ne e heless, he p esen s udy e eals ha di e en de elopmen pa ame e s can be
di e en ly in luenced by ex eme d ough , e en hough hese only las ed o a ound hal o
he du a ion o hei la al de elopmen . Fu he mo e we show ha plan communi y
composi ion (and he e o e also compe i ion and he p esence o speci ic plan unc ional
ai s) al e s lea me abolism and he eby a ec s he bi o es. Conce ning he deba e on he
hypo heses on plan -s ess (Whi e1984) e sus plan - igou (P ice 1991) we con i m o he
s udies which show he dependence o he bi o e pe o mance on plan -s ess le el (Schei s
and de B uyn 2005), in es iga ed ai s (Co nelissen e al. 2008) and insec - eeding guild
(La sson 1989; Ko iche a e al. 1998; Hube y and Denno 2004), as ou esul s di e om
o he d ough expe imen s using di e en eeding guilds (EnglishLoeb e al. 1997; de B uyn
e al. 2002). Di e ences o o he s udies may also be a ibu ed o ou speci ic expe imen al
condi ions: Plan s we e g own in expe imen al communi ies in he ield, no in isola ed po s in
he g eenhouse. This ensu es mo e ealis ic plan g ow h condi ions. In addi ion, he
he bi o es in ou s udy ha e no been g own on he plan s, bu we e ed in clima e chambe s,
o mo e clea ly ela e he ob ained esul s o changes in plan me aboli es. Ano he eason o
di e ences o o he s udies and p ominen hypo heses migh be ha he plan s we e se e ly
s essed, bu also eleased om s ess and eco e ed du ing he eeding expe imen .
Acco ding o Hube y and Denno (2004) his migh ha e caused di e ences in he bi o e
pe o mance when compa ed o eeding expe imen s unde cons an s ess. Ou s udy
p o ides addi ional e idence ha he ni ogen limi a ion hypo hesis, s a ing ha high ni ogen
con en s a e bene icial o he bi o e pe o mance (Whi e1984) does no seem o be widely
applicable o e all de elopmen ai s and s ages (Fische and Fiedle 2000). Simila o
Fische & Fiedle (2000) ou da a p o ide hin s ha highe p o ein o ni ogen con en s a e
bene icial o a as e a e o de elopmen and highe la al su i al, al hough hey migh
educe adul eclosion.
Conclusion
Ou indings sugges ha ex eme d ough s, which a e p ojec ed o inc ease in equency wi h
clima e change, can also a ec he de elopmen o he bi o es. This is p ima ily caused by a
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
93
Impo an aspec s a e no only he pe sis ence o species and subs a e, bu also he
possibili ies o species o ecolonize (Nys om and Folke, 2001). An o e iew on he a ious
unde s andings o ecological memo y is p o ided by Golinski e al. (2008). The complexi y o
he p ocesses in ol ed in he b oad de ini ion o ecological memo y including empo al
pa e ns up o e olu iona y scales and spa ial pa e ns up o landscape le els (Thompson e al.,
2001), may limi i s applicabili y in ecology as well as he possibili y o assess and measu e
such a memo y. I hus appea s necessa y o apply a educ ionis amewo k o os e ou
unde s anding o he impo ance o ecological memo y in imes o global change (see Table 1
o examples and le els o ecological memo y a e dis u bances). In he ollowing, we ocus
on ecological s ess memo y o single indi iduals as a s a ing poin .
Ecological s ess memo y is de ined he e as any esponse o a single plan a e a
s ess expe ience ha imp o es he esponse o he plan owa ds u u e s ess expe ience and
which is assessed on a whole plan le el (Fig. 1).
nega i e esponse
neu al esponse
imp o ed esponse
no s ess no s ess
s ess s ess
acclima ion
eco e y
s ess memo y
eco e y acclima ion
s ess memo y
Æimp o ed
pe o mance
damage lagged s ess
e ec s
collapse
esponse o
s ess
ime
nega i e esponse
neu al esponse
imp o ed esponse
no s ess no s ess
s ess s ess
acclima ion
eco e y
s ess memo y
eco e y acclima ion
s ess memo y
Æimp o ed
pe o mance
damage lagged s ess
e ec s
collapse
esponse o
s ess
ime
Fig.1 Plan s ess esponse unde single and epea ed s ess wi hou acclima ion leads o s ess damage
(nega i e esponse), exhibi ing acclima ion (neu al esponse) and exhibi ing an addi ional ecological
s ess memo y (imp o ed esponse). Acclima ion helps o p e en s ess damage and o p omo e eco e y,
despi e o en leading o educed g ow h du ing s ess. An ecological s ess memo y exis s, when he plan
keeps a so o “s ess imp in ” a e s ess exposu e ha imp o es plan esponse o ecu en s ess
compa ed o plan s wi hou s ess memo y. Lagged s ess e ec s a e de imen al e ec s ha occu some
ime a e he s ess occu ed. S ess damage may lead o e en g ea e damage o comple e collapse when
ecu en s ess is applied

Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
94
An ecological s ess memo y migh in ol e he pe sis ence o acclima ion mechanisms
and p o ec i e subs ances. Howe e , i is no acclima ion pe se, as such a memo y emains
ac i e long a e he s ess has been applied, and enables he plan o espond quicke and
mo e adequa e o a ecu en s ess e en . I hus equi es a pe sis ing “imp in ” modi ying
u u e s ess esponse. Fo acclima ion, he plan does no need o expe ience eal s ess, as,
e.g. o os acclima ion, ce ain en i onmen al cues a e su icien o igge acclima ion
wi hin he seasonal li e cycle pe o mance. Ecological s ess memo y has a empo al
dimension and, in his sense can only be s udied a e he s ess s opped and he plan
eco e ed, e.g. ook up i s p e-s ess me abolism again and epai ed o compensa ed damage.
A e applying ecu en s ess, ecological s ess memo y should lead o an imp o ed
pe o mance when compa ed o plan s wi hou a pe sis ing s ess memo y.
Con as ingly, lagged o delayed s ess e ec s a e de imen al e ec s o single s ess
e en s ha become clea ly appa en only a e some ime, e.g. when he plan dies o a
communi y collapses (Fig. 1). The esponse o ees o d ough , o ins ance, is o en
exp essed in inc eased mo ali y bu his may happen e en se e al yea s a e he d ough
e en (Bigle e al., 2007). Likewise, al e a ions in soil os e en s can lead o inc eased
mo ali y o dwa -sh ubs a e mo e han one yea , bu wi h no appa en e ec s in he i s
yea a e he s ess e en (K eyling e al., 2010). Such lagged esponses clea ly indica e
ca y-o e e ec s in i ness which a e no easily de ec able di ec ly a e he e en and which
may explain he indings o educed esilience upon epea ed s ess e en s. Fo example,
Llo e e al. (2004) in es iga ed he impac o ecu en d ough (1985 and 1995) on
esp ou ing and die-back in Que cus ilex. They ound a p og essi e loss o indi idual
esilience upon ecu en d ough , as he abili y o su i e and esp ou was educed compa ed
o he i s d ough . Muelle e al. (2005) examined Pinus edulis and Junipe us monospe ma
mo ali y a e ex eme d ough episodes and also ound a educ ion o esilience and a highe
mo ali y a e o a ecu en d ough in 2002 compa ed o he p e ious d ough in 1996. I
may ha e been ha de imen al e ec s ha e pe sis ed e en a e many yea s and plan s may
no ha e been eco e ed be o e he ollowing s ess exposu e: S a ch s ocks in ligno ube s o
Q. ilex we e ound no o be es o ed o hei p e-s ess alues e en 10 yea s a e an ex eme
d ough (Lopez e al., 2009). Thus, o clea ly dis inguish such lagged s ess e ec s om an
ecological s ess memo y i may be necessa y o expe imen s o apply a ecu en s ess e en
and o compa e he answe o ecu en ly s essed plan s o single s essed plan s. Only when
he pe o mance o ecu en s ess is imp o ed when compa ed o single s essed plan s, he
de ini ion allows calling his an ecological s ess memo y.
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
95
In he ollowing, we e iew mechanisms o d ough ole ance and e idences o
d ough memo y, o os ole ance and os memo y as well as o hea s ess ole ance and
hea s ess memo y.
3. D ough ole ance and d ough memo y
Plan s a e able o acclima e o d ough s ess, he eby inc easing hei d ough
ole ance. Mechanisms o acclima ion include he accumula ion o osmop o ec i e p o eins,
like dehyd ins (Bohne , 2000; Lambe s e al., 2008), he accumula ion o soluble suga s
(Lambe s e al., 2008; Wal e e al., 2012), a educ ion o he pho osyn he ic appa a us along
wi h addi ional mechanisms o p e en damage by eac i e oxygen species (Munne-Bosch
and Aleg e, 2000) and he accumula ion o compa ible solu es (p oline, be aine) (Bohne ,
2000). Changes in gene exp ession ha accompany d ough acclima ion a e o en ABA-
media ed, and up egula ed genes include genes o he LEA amily (la e emb yogenesis
abundan ) (Bohne , 2000; Lambe s e al., 2008). In addi ion o physiological changes,
pheno ypic and mo phological esponses can be ini ia ed du ing d ough s ess, such as an
inc eased oo o shoo a io o he de elopmen o oo s in deepe soil laye s (Newman e al.,
2006).
Some ecen indings indica e he exis ence o an ecological d ough memo y: Wal e
e al. (2011) ound an inc ease in pho op o ec ion in single g ass plan s unde epea ed
d ough when compa ed o plan s ha we e no subjec ed o d ough p e iously, e en se e al
weeks a e he i s d ough was applied and a e he plan s we e comple ely cu and eg own
(Fig. 2A).
Simila ly, Ona e e al. (2011) showed ha U ica dioica subjec ed o combined
d ough and nu ien de iciency in hei ju enile phase e ealed imp o ed d ough s ess
ole ance in ma u e lea es, especially in ep oduc i e shoo s.
Unde labo a o y condi ions, Goh e al. (2003) ound ha A abidopsis haliana
epea edly subjec ed o high le els o abscisic acid (ABA), also in ol ed in d ough s ess
signaling and esponse, led o a o ma ion o ecological s ess memo y, as gene exp ession
was changed in esponse o ollowing s ess e en s compa ed o non- ea ed plan s. Knigh e
al. (1998) obse ed changes in d ough s ess-induced calcium-signaling a e plan s had
encoun e ed ei he osmo ic o oxida i e s ess p e iously.
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
96
Fig. 2 Examples o (A) ecological d ough memo y (adap ed om Wal e e al. 2011), (B) ecological hea
memo y (adap ed om Whi le e al. 2009) and (C) o an ecological c oss-s ess memo y (adap ed om
K eyling e al. 2012b). Plan s we e ei he uns essed (ligh g ay ba s) o subjec ed o d ough s ess (A, C)
o hea s ess (B) (da k g ay ba s; p e ea men indica ed on he y-axis) and pe o mance was measu ed
unde subsequen d ough (A), hea (B) o os (C) s ess (as indica ed by inse s) in he same plan s (A,
C) o in he F 3 gene a ion (B). Di e en le e s indica e signi ican di e ences be ween plan s wi h and
wi hou p e-s ess acco ding o he sou ces
Cuk e al. (2010) showed ha modi ied ac i i y o an ioxida i e enzymes (ca alase,
asco ba e pe oxidase), which a e o en also up egula ed unde d ough , is inhe i ed o he nex
gene a ion o A. haliana. I seems likely ha ma e nal plan s inhe i ing s ess ole ance would
e eal an ecological s ess memo y hemsel es.
To sum up, he e is e idence ha ce ain physiological p ocesses in plan s a e
modi ied by o me s ess e en s. These modi ica ions can be decisi e in ace o epea ed
e en s and may s imula e a as e s a o p o ec i e mechanisms and inc eased s ess
ole ance and compensa ion. Howe e , s udies in es iga ing his opic a e a e and mo e
s udies in es iga ing d ough memo y in di e en species and also unde mo e na u al
condi ions and s udies compa ing he esponse o ecu en d ough manipula ions o he
esponse o single d ough manipula ions a e needed in he u u e.
50
Seed numbe pe plan
P and F1 23°C
P and F1 30°C
F3: hea
500
1500
a
b
(B)
Li ing biomass [%]
0
10
20
30
40
50
60
70
a
b
d ough
No p e iousd ough
P e iousd ough
(A)
a
b
No p e iousd ough
P e iousd ough
os
Cold ha diness [LT50 °C]
-40
0
-10
-20
-30
(C)
50
Seed numbe pe plan
P and F1 23°C
P and F1 30°C
F3: hea
500
1500
a
b
(B)
50
Seed numbe pe plan
P and F1 23°C
P and F1 30°C
F3: hea
500
1500
a
b
(B)
Li ing biomass [%]
0
10
20
30
40
50
60
70
a
b
d ough
No p e iousd ough
P e iousd ough
(A)
Li ing biomass [%]
0
10
20
30
40
50
60
70
a
b
d ough
No p e iousd ough
P e iousd ough
(A)
a
b
No p e iousd ough
P e iousd ough
os
Cold ha diness [LT50 °C]
-40
0
-10
-20
-30
(C)
a
b
No p e iousd ough
P e iousd ough
os
Cold ha diness [LT50 °C]
-40
0
-10
-20
-30
a
b
No p e iousd ough
P e iousd ough
os
Cold ha diness [LT50 °C]
-40
0
-10
-20
-30
(C)
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
97
4. F os ole ance and os memo y
In egions whe e subze o empe a u es a e eached, pe ennial plan s show he po en ial
o acclima e o os o educe os damage, caused by in acellula ice c ys als and
dehyd a ion. As apoplas ic ice o ma ion leads o cell dehyd a ion, d ough acclima ion and
os acclima ion o en in ol e he same mechanisms, like accumula ion o soluble suga s o
ansc ip ion o dehyd ins and LEA-genes (Lambe s e al., 2008; Janska e al., 2010). F os
acclima ion is igge ed by low empe a u e and by changes in he pho ope iod (Thomashow,
1999; Janska e al., 2010). Ha dening usually akes se e al weeks, while deha dening, i.e. he
loss o os ha diness, can occu wi hin hou s a e empe a u e inc ease (Rapacz e al.,
2000), lea ing he plan s ulne able o sho - e m la e os e en s du ing he g owing season
o a e win e wa ming e en s.
The equency o os days and nigh s is expec ed o dec ease in a ious biomes
unde global clima e wa ming (IPCC 2007), ye , an inc ease in minimum empe a u e o e
win e is unlikely (Kod a e al., 2011). Obse ed and p ojec ed educ ion in snow co e ,
which ac s as insula ion o many plan s, in combina ion wi h mo e a iable ai empe a u es
may u he exace ba e he equency o os s ess in many no he n egions (K eyling,
2010). As global wa ming may egionally lead o an ea lie deha dening and onse o he
g owing season, he isk o la e os damage is likely o inc ease, when he iming o la e
os e en s is no changing (Rigby and Po po a o, 2008;Woldendo p e al., 2008).
Fu he mo e, global wa ming migh lead o mo e equen eeze- haw cycles du ing win e ,
possibly associa ed wi h (pa ial) deha dening a e especially wa m win e days, leading o
os damage a u he sub-ze o empe a u es (Bokho s e al., 2009).
I is well es ablished ha plan s a e able o “ emembe “ low empe a u es o e a
ce ain ime span, as e naliza ion, he p omo ion o sp ing lowe ing a o ed by low
empe a u es equi es some so o win e -memo y (Sung and Amasino, 2005). He e, we
a gue ha cold ole ance acclima ion is no ecological s ess memo y i sel , as he plan s
ha den unde low empe a u es bu wi hou expe iencing os s ess. Cold acclima ion a he
is an e olu iona y esponse o a oid os s ess. Expe ience o os s ess, o example du ing
eeze- haw cycles o unde la e os e en s, could enable he plan o eac di e en ly o he
nex os spell, e en wi hou p io acclima ion.
Tahkoko pi e al. (2007) ound inc eased an hocyanin le els in sp ing in newly g own
s ems o Vaccinium my illus a e plan s had been subjec ed o os s ess in win e ,
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
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compa ed o plan s no expe iencing os s ess be o e. This s ongly indica es an ecological
os s ess memo y on a whole plan le el.
Unde labo a o y condi ions, Knigh e al. (1996) ound a modi ied calcium signa u e
in A. haliana a e plan s had expe ienced a cold shock be o e. Calcium ac s as a second
messenge in low empe a u e signaling and may he e o e igge al e ed esponse o epea ed
os .
Howe e , Polle e al. (1996) showed ha sp uce needles su i ing a sp ing os e en
e ealed less an ioxida i e enzymes and pigmen s in he ollowing all, hus p obably
showing educed os p o ec ion despi e a ansien inc ease in an ioxida i e enzymes a e
sp ing os was applied. This shows ha os s ess may lead o a loss o esilience a he
han o he o ma ion o a “posi i e” ecological os memo y.
To sum up, ew s udies hin owa ds he exis ence o an ecological os s ess memo y
(Tahkoko pi e al. 2007; Knigh e al. 1996), al hough he esponse o epea ed os s ess has
no ye been in es iga ed. I emains o be elucida ed i os s ess expe ience helps o su i e
subsequen os s ess, as one s udy also indica es a dec ease in os esis ance a e os
s ess (Polle e al. 1996).
5. Hea s ess ole ance and hea s ess memo y
On a cell-le el, hea s ess acclima ion is a he well unde s ood: Upon exposu e o
ex emely high empe a u es, exp ession o no mal housekeeping genes is s opped and hea
shock p o eins (HSP), which ac o p e en p o ein damage o pho o-oxida ion and which
epai al eady dena u a ed p o eins (chape ones) a e inc easingly syn hesized (Pa cellie e al.,
2003; Baniwal e al., 2004; Ko ak e al., 2007). Fu he mo e, compa ible solu es like p olin o
be aine ac o s abilize p o eins (Schulze e al., 2005).
Unde global wa ming i is e y likely ha hea wa es will inc ease in equency and
magni ude o e mos land a eas, as indica ed, e.g., by he Eu opean mega-hea wa es in 2003
and 2010 (Schä e al., 2004; Ba ioped o e al., 2011; IPCC 2011). Ye , s udies in es iga ing
an ecological hea s ess memo y a e widely lacking in plan ecology.
In e es ingly, a ansgene a ional ecological s ess memo y was ound o mild hea
s ess: Plan s om he F3 gene a ion showed a hea -speci ic i ness imp o emen when he
pa en al and F1 gene a ion had been ea ed wi h mild hea (30°C), e en when he F2
gene a ion was g own unde no mal condi ions (Whi le e al., 2009) (Fig. 2B). As o all

Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
99
ansgene a ional s udies, he exis ence o ecological s ess memo y wi hin one plan
gene a ion was no es ed, bu is implied by he inhe i ance o s ess ole ance.
To conclude, we ound no s udy in es iga ing an ecological hea s ess memo y in
single plan s. Ne e heless, he esul s by Whi le e al. (2009) indica e he exis ence o a hea
s ess memo y imp o ing hea ole ance a e hea s ess was al eady expe ienced in he pas .
6. C oss-s ess memo y
As os , hea and d ough s ess all in ol e cell dehyd a ion, acclima ion mechanisms
a e pa ly he same (Beck e al., 2007). I is hus possible ha acclima ion and o ma ion o a
s ess memo y o one kind o s ess also p e en s damage by o he s esso s, p o iding c oss-
s ess memo y and ole ance. Fo ins ance, os ole ance o local popula ions o eco ypes,
espec i ely, is ela ed o d ough ole ance (Blodne e al., 2005).
Mo e speci ically, exposu e o an ex eme d ough e en in he p eceding yea was
ound o suppo la e os ole ance in g ass species (K eyling e al., 2012a) and maximum
os ha diness in ju enile Pinus nig a (K eyling e al., 2012b) (Fig. 2C).
Ano he o m o c oss-s ess ole ance is inc eased he bi o e esis ance a e he
expe ience o abio ic s ess like d ough , caused by an inc ease in C-based seconda y
me aboli es upon g ow h es ic ion due o abio ic s ess (He ms and Ma son, 1992). I is
unclea ye , how long such modi ica ions o seconda y compounds a e main ained. Howe e ,
Ag awal (2002) showed ha he p ogeny o ma e nal plan s a acked by he bi o es also
e ealed highe induced esis ance owa ds he bi o es. This implies he i abili y and hus a
o m o s ess memo y o induced de ense, al hough i is unclea i his could also ac o
p e en d ough , hea o os damage.
7. Possible mechanisms behind an ecological s ess memo y
As possible mechanisms o an ecological s ess memo y, B uce e al. (2007) sugges
he accumula ion o ansc ip ion ac o s o p o eins o acili a e a as esponse upon epea ed
s ess exposu e as well as epigene ic mechanisms, such as his one modi ica ions o chemical
changes a he DNA (me hyla ion, ace yla ion) ha a e inhe i ed h ough mi o ic o e en
meio ic cell di isions (Bossdo e al., 2008; Chinnusamy e al., 2008; Boyko and Ko alchuk,
2011). Ano he possibili y is he accumula ion o p o ec i e subs ances. Howe e , his is no
likely o be e y impo an , as syn hesis o p o ec i e subs ances is cos ly and o en p e en s
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
ex emes
100
no mal g ow h (He ms and Ma son, 1992). Today, i is well es ablished ha plan s ess
induces epigene ic changes (Goh e al., 2003; Chinnusamy e al., 2008). I was shown ha
epigene ic changes upon UV-C and lagellin exposu e (Molinie e al., 2006), upon TMV
( obacco-mosaic- i us)-exposu e (Boyko e al., 2007), upon pa hogen o he bi o e a ack o
low nu ien s (Ve hoe en e al., 2010) and upon ni ogen de iciency (Kou e al., 2011) a e
inhe i ed. Fu he mo e, inhe i ed DNA-hypome hyla ion in ice seedlings inc eased pa hogen
esis ance (Akimo o e al., 2007) and inhe i ed epigene ic changes upon TMV exposu e
inc eased pa hogen esis ance in p ogenies (Ka hi ia e al., 2010) Howe e , no gene al
he i abili y o epigene ic changes upon s ess exposu e was ound in A. haliana, implying
ha ansgene a ional epigene ic memo y seems o be es ic ed o special condi ions (Pecinka
e al., 2009). The esul s o Tahkoko pi e al. (2007) hin owa ds epigene ic changes as
unde lying mechanisms o an ecological os memo y, as he new s ems g owing in sp ing
ne e expe ienced os s ess hemsel es bu e ealed modi ica ions (see Sec ion 4). Thus,
in o ma ion had o be con eyed h ough mi o ic di isions.
A u he possibili y o e ain an ecological s ess memo y a e changes in phenology o
mo phology o he plan ha emain s able o e longe ime scales han me e changes in he
accumula ion o p o ec i e subs ances. Shi s in oo o shoo a io in esponse o d ough
(Kalapos e al., 1996; Kahmen e al., 2005) o win e wa ming pulses (K eyling e al., 2008)
a e one ob ious mo phological esponse wi h implica ions o u u e d ough ole ance.
Fu he mo e, speci ic lea a ea can be adap ed o d ough condi ions (Kalapos e al., 1996),
he eby educing wa e loss o his issue also o u u e d ough e en s (see Table 1 o
examples o mechanisms o an ecological s ess memo y).
8. Resea ch challenges
S udies in es iga ing ecological s ess memo y a e a e. Mos s udies on he du a ion
and he i abili y o plan s ess a e conduc ed on a cellula le el and ocus on gene ic o
epigene ic aspec s. In such s udies, ime spans be ween he ini ial and he epea ed s ess is
usually es ic ed o only se e al hou s o days (B uce e al., 2007). Mo e ecologically
ele an esea ch and assessmen s o s ess ole ance and ecological s ess memo y a e
needed. Fu he mo e, mul igene a ional epigene ic s udies should conside consequences o
ex eme wea he e en s mo e p ominen ly.
In o de o e alua e he ecological ele ance o ecological s ess memo y, assessing
ecologically meaning ul pa ame e s a he plan le el in con olled lab expe imen s needs o
Manusc ip 3: Ecological s ess memo y and c oss s ess ole ance in plan s in he ace o clima e
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101
be accompanied by ield expe imen s and obse a ions a e na u ally occu ing ex emes.
He e, long- e m ield- and moni o ing s udies in es iga ing he esponse o na u ally occu ing
ex emes in na u al plan communi ies migh be e y aluable. To simul aneously elucida e
unde lying p ocesses and mechanisms, coope a ion be ween ecologis s and molecula
biologis s a e u gen ly needed. Fo ins ance, i is s ill unclea whe he unseasonable os
damage in sp ing in luences acclima ion in he ollowing all and i his migh be de imen al
o bene icial.
In pa icula he absolu e deg ee and he empo al s abili y o ecological s ess memo y
equi es a en ion because ecological s ess memo y could play an impo an ole in
s abilizing ecological communi ies in he ace o mo e equen ex eme clima ic e en s. He e,
we sugges ha ecological s ess memo y can, a leas pa ly, explain he su p isingly weak
e ec s o epea ed ex eme d ough e en s on he p oduc i i y o g assland communi ies
(Jen sch e al. 2011). He i abili y o bene icial s ess memo y o ollowing gene a ions,
po en ially by epigene ic p ocesses, is ano he highly ele an aspec o ou unde s anding o
ecological esponse o mo e equen ex eme clima ic e en s.
To conclude, he e a e e idences o he exis ence o an ecological s ess memo y.
Howe e , mechanisms and consequences a e no ye well in es iga ed. A s ess memo y o
single plan s migh ac o s abilize plan communi ies unde equen clima ic ex emes and
migh inc ease esilience. I migh be e en possible o mi iga e de imen al e ec s o ex eme
e en s by a i icially applying milde s ess on a small scale, e.g. o ag icul u e. Howe e , i
is no ye clea i , on a ield and landscape scale, lagged de imen al e ec s migh lead o a
educ ion o esilience unde epea ed ex eme wea he e en s ha ou weigh possible posi i e
e ec s o an ecological s ess memo y. Fu he mo e, inc eased acclima ion owa ds ecu ing
s esso s migh educe mo ali y o plan s, bu migh as well educe ag icul u al yield i plan s
educe hei pho osyn he ic ac i i y o p e en damage. Hope ully, u u e esea ch will
con ibu e o elucida e mechanisms and consequences o an ecological s ess memo y.
Acknowledgemen s: We hank wo anonymous e iewe s o aluable hin s and commen s
o cla i y ou concep s and imp o e ou manusc ip .
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Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
109
Ge many (49°55´19´´N,11°3455``E, 365 m asl). Tubes we e embedded in o he homogenized
soil (loamy sand consis ing o 82 % sand, 13 % sil , 5 % clay o a dep h o 80cm). Plan s we e
kep unde na u al ambien condi ions o wo yea s and ha es ed wice pe yea .
Fu he mo e, ubes we e pe iodically weeded. In Ap il 2009, ubes, including he soil, we e
a anged unde a ain-ou -shel e o he expe imen . To a oid he la e al low o wa e in o
he po s, hey we e placed on pla es on a plas ic shee . The anspa en ain-ou shel e was
le open a he side up o 80 cm, allowing ai exchange nea o he su ace and hus a oiding
g eenhouse-e ec s. Plan s we e subjec ed o wo di e en ea men s and a anged in a
comple ely andomized design: 28 plan s in he ecu en d ough ea men ( wo plan s died
in he wo yea s be o e he expe imen s a ed) we e subjec ed o an ea ly d ough in June
2009 (D1), whe eby wa e was comple ely wi hheld o 16 days om June 3 d un il June 18 h.
The same plan s we e subjec ed o a la e d ough (D2), whe eby wa e was wi hheld o 16
days om Sep embe 4 h o Sep embe 19 h. The ecu en d ough ea men was compa ed o
a single d ough ea men : 27 eplica es ( h ee plan s died p io o he expe imen ) we e
wa e ed egula ly e e y hi d day wi h 300ml ain wa e (C1) while he i s d ough pe iod
was applied o double-s essed plan s. C1 plan s we e exposed o hei i s d ough in
Sep embe (C2), concomi an o he second d ough o he ecu en d ough ea men (See
Fig. 1 o an o e iew).
Fig. 1 O e iew on he expe imen al ime cou se and on applied
ea men s. In June, d ough was applied o 16 days o D1 plan s,
while C1 plan s we e wa e ed egula ly. All plan s we e wa e ed
a e he 16 h day un il he onse o he la e d ough in Sep embe .
In Sep embe all plan s (D2 and C2) we e subjec ed o he
d ough o 16 days and we e wa e ed a e ha .
Only in compa ing plan s subjec ed o a second, la e d ough (D2) o plan s
expe iencing hei i s d ough (C2) a he same poin in ime, we could p e en con ounding
o po en ial d ough memo y e ec s wi h seasonali y o iming e ec s. A compa ison be ween
he esponse o he i s d ough in June and he esponse o he second d ough in Sep embe
is hus no alid o in es iga e po en ial d ough memo y e ec s. We also did no ha e a well-
wa e ed con ol in Sep embe , as we we e in e es ed in a po en ial “d ough -memo y”, which
can only be in es iga ed by compa ing single-s essed wi h double-s essed plan s. To
quan i y e ec s o a single, ea ly d ough (D1), d ough plan s we e compa ed o well-
wa e ed plan s du ing he i s d ough pe iod (C1). All plan s we e wa e ed wi h he same
June 3 d-18 h Sep . 4 h-19 h
D1
C1
D2
C2
d ough ed
wa e ed
June 3 d-18 h Sep . 4 h-19 h
D1
C1
D2
C2
d ough ed
wa e ed

Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
110
amoun o wa e (300 ml, e e y hi d day) un il he onse o he expe imen and in be ween
he wo d ough ea men s.
2.2. Abo eg ound biomass
Abo eg ound biomass was ha es ed on July 5 h, 17 days a e he i s d ough (D1
and C1) ended, and on Oc obe 9 h, 21 days a e he second d ough ended (D2 and C2). A e
his ime, e e sible d ough damages should ha e been eco e ed. As we only compa e D2
wi h C2 plan s and D1 wi h C1 plan s, he di e ence o ou days in eco e y ime a e
d ough does no ha e an e ec on he esul s. Plan s we e cu 4 cm abo e he g ound in o de
o simula e common managemen echniques in meadows, so ed in o li ing (g een) and dead
biomass. Dead biomass was de ined as wil ed, b own plan pa s ha los chlo ophyll.
Biomass was d ied a 70° C o 72 hou s and weighed. Pe cen age o dead biomass was
calcula ed as pe cen age o o en-d ied, dead biomass in ela ion o o e all o en-d ied biomass
o indi idual plan s.
2.3. Rela i e lea wa e con en (RWC)
Rela i e lea wa e con en was de e mined in he a e noon o he 13 h day o he i s
and second d ough ea men (June 15 h and Sep embe 16 h), acco ding o Ba s and
Wea he ley (1962).The second lowes lea o each plan was cu , s o ed in a mois ened plas ic
bag o anspo , and immedia ely weighed o de e mine esh weigh (FW). Lea es we e
s o ed in dis illed wa e a 4° C o e nigh and weighed he nex mo ning o de e mine u gid
weigh (TW). A e wa ds lea es we e d ied a 70° C and he d y weigh (DW) was
de e mined.
RWC was calcula ed as:
100*
)(
)(
(%) DWTW
DWFW
RWC −
−
=
2.4. Chlo ophyll a luo escence
Chlo ophyll a luo escence was eco ded using a pulse-ampli ude-modula ed
pho osyn hesis yield analyze (PAM 2000 and Mini-PAM) (Wal z, E el ich, Ge many) wi h
a lea clip holde as desc ibed by Bilge e al. (1995). The second o hi d ully-expanded
lea es we e measu ed on ou di e en blades o one indi idual. Fou measu emen s pe plan
we e a e aged o u he analysis. We ob ained p edawn luo escence alues (be ween 2:00
and 4:00) a he end o he i s d ough ea men , h oughou he second d ough pe iod and
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
111
h oughou he ea ly eco e y phase a e he second d ough . The maximum quan um
e iciency o pho osys em II was calcula ed as F /Fm. Va iable luo escence (F ) and
maximum luo escence (Fm) we e measu ed be o e dawn. F was calcula ed as Fm-F0, Fm
being he maximum luo escence o he da k adap ed lea a e applying a sa u a ing ligh
pulse and F0 being he s eady s a e luo escence yield o he da k adap ed lea (Maxwell and
Johnson, 2000).
To enable a compa ison be ween absolu e luo escence alues, a luo escence s anda d
ma e ial was measu ed be o e and a e each measu ing cycle. S anda d measu emen s we e
used o no malize he luo escence alues ob ained and o calib a e he wo di e en PAMs in
use. P edawn measu emen s o luo escence a he da k adap ed lea allow d awing
conclusions abou unde lying p ocesses which al e plan pho osyn he ic pe o mance and
abou pho oinhibi o y damage and non-pho ochemical quenching (Maxwell and Johnson,
2000). Absolu e F0 and Fm alues we e aken o sepa a e he e ec s o pho odamage,
becoming appa en wi h an inc ease o F0, om he e ec s o pho op o ec ion ela ed o
enhanced non-pho ochemical quenching, becoming appa en wi h a dec ease in Fm (Osmond
e al., 1993; A aus e al., 1998; Maxwell and Johnson, 2000).
2.5. Lea gas exchange
The ne CO2 assimila ion a e (Pn) and anspi a ion we e measu ed a midday
(be ween 11:30 and 13:30), when d ough s ess should be a i s maximum, due o high
empe a u e and i adiance. I was measu ed on he second, ully de eloped lea o each plan
using a gas-exchange sys em (Li-6400, Li-Co , Lincoln, NE, USA) equipped wi h a CO2
ca idge o adjus and main ain cons an CO2 o 400 μmol mol–1 ai wi hin he lea cu e e.
Gas exchange measu emen s we e conduc ed on clea days wi hou clouds o main ain
cons an PPFD. A e eaching s eady-s a e pho osyn hesis, da a we e logged. The lea a ea
was es ima ed simul aneously by measu ing he lea wid h and la e on used o co ec alues
o ne pho osyn hesis, as lea blades did no ill he whole lea cu e e. Gas exchange was
measu ed unde ambien ligh condi ions a he end o he i s d ough pe iod, in he ea ly and
la e d ough pe iod and in he ea ly eco e y phase a e he d ough .
2.6. S a is ical analysis
To de e mine signi ican di e ences be ween single and ecu en d ough ea men s,
analyses o a iance we e pe o med o all a iables o each sampling da e. We de ined
“ ea men ” as a ixed ac o . “P o enance” was a andom ac o in his expe imen , as
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
112
p o enances we e chosen andomly ou o a la ge popula ion o p o enances, and as we we e
no in e es ed in he speci ic p o enances, bu in he whole popula ion o ou plan s (Do mann
and Kühn, 2009). We examined he esiduals agains i ed plo s and no mal qq-plo s p io o
each analysis o es whe he he assump ions o ANOVA, homogenei y o a iances and
no mali y, could be me (Fa away, 2006). I his was no he case, da a we e
powe ans o med ( luo escence da a), log- ans o med (absolu e biomass da a) o a csin-
ans o med ( ela i e wa e con en ) acco dingly.
All s a is ical analyses we e pe o med using R 2.11.0 (R De elopmen Co e Team 2010). Fo
mixed e ec models we used he so wa e package nlme (Pinhei o e al., 2008).
3. Resul s
3.1. E ec s o he i s d ough (D1)
Tempe a u e [°C]
10
12
14
16
18
20
22
24
26
28
0
2
4
6
8
10
12
14
P ecipi a ion [mm]
0 5 10 15 20
8
10
12
14
16
18
20
22
24
26
A e age Tempe a u e
Maximum Tempe a u e
Day o expe imen
0
5
10
15
P ecipi a ion
(a)
(b)
Tempe a u e [°C]
10
12
14
16
18
20
22
24
26
28
0
2
4
6
8
10
12
14
P ecipi a ion [mm]
0 5 10 15 20
8
10
12
14
16
18
20
22
24
26
A e age Tempe a u e
Maximum Tempe a u e
Day o expe imen
0
5
10
15
P ecipi a ion
(a)
(b)
Tempe a u e [°C]
10
12
14
16
18
20
22
24
26
28
0
2
4
6
8
10
12
14
P ecipi a ion [mm]
0 5 10 15 20
8
10
12
14
16
18
20
22
24
26
A e age Tempe a u e
Maximum Tempe a u e
A e age Tempe a u e
Maximum Tempe a u e
Day o expe imen
0
5
10
15
P ecipi a ion
(a)
(b)
Fig. 2 Cou se o daily maximum empe a u e (g ey squa es), daily a e age empe a u e (black ci cles)
and p ecipi a ion (da k g ey ba s) a he s udy si e du ing he expe imen al pe iods in June (a) and
Sep embe (b). Ve ical black line indica es he s a o he d ough ea men , g ey dashed line indica es
when ewa e ing s a ed.
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
113
Tempe a u e and p ecipi a ion da a du ing he expe imen al pe iod in June a e shown
in Figu e 2.
Ea ly d ough ea men (D1) in June signi ican ly educed he ela i e lea wa e
con en measu ed a he end o he d ough ea men compa ed o he well-wa e ed con ol
(C1) by a ound 22% (P<0.001; Fig. 3a).
C1 D1
Rela i e lea wa e con en [%]
020
40 60 80 100
(a)
Pn [µmol CO
2
m
-2
s
-1
]
02 4 6 8 10 12
(d)
C1 D1
F /Fm
0.0 0.2 0.4 0.6 0.8 1.0
(c)
C1 D1 C1 D1
Li ing biomass [%]
0204060
80
(b)
C1 D1
Rela i e lea wa e con en [%]
020
40 60 80 100
(a)
Pn [µmol CO
2
m
-2
s
-1
]
02 4 6 8 10 12
(d)
C1 D1
F /Fm
0.0 0.2 0.4 0.6 0.8 1.0
(c)
C1 D1 C1 D1
Li ing biomass [%]
0204060
80
(b)
Fig. 3 E ec s o he i s d ough (D1) in June (da k g ey) compa ed o well-wa e ed con ol plan s (C1)
(ligh g ey) on ela i e wa e con en o lea es (a), pe cen age o li ing biomass (b), maximum quan um
e iciency F /Fm, measu ed p edawn (c), and ne pho osyn hesis Pn, measu ed du ing midday (d).
Rela i e lea wa e con en (a) was measu ed h ee days be o e he end o he d ough ea men ,
maximum quan um e iciency F /Fm (c) and ne pho osyn hesis Pn (d) we e eco ded on he las day o
he d ough ea men . To al abo eg ound ha es was conduc ed 17 days a e he end o he i s
d ough ea men and he pe cen age o dead biomass (d y weigh ) (b) was calcula ed. Means and SE a e
shown, as e isks indica e signi icance (*, P≤ 0.05; **, P≤ 0.01; ***, P≤ 0.001).
Ea ly d ough ea men (D1) had no e ec on he o al abo eg ound biomass when
compa ed wi h he well-wa e ed con ol ea men (C1) (da a no shown). Howe e , d ough
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
114
educed he pe cen age o li ing biomass signi ican ly by a ound 15% (P=0.03; Fig. 3b). On
he las day o ea ly d ough ea men (D1) he pho ochemical e iciency (F /Fm) o plan s
unde d ough was signi ican ly educed compa ed o he well-wa e ed con ol (P=0.004; Fig.
3c).
Plan s unde ea ly d ough ea men (D1) exhibi ed educed ne pho osyn hesis by
58% on he las day o he ea ly d ough ea men (P<0.001; Fig. 3d).
3.2. E ec s o ecu en d ough e en s (D2) compa ed o a single d ough e en (C2) in
Sep embe
3.2.1. Tempe a u e, Rela i e lea wa e con en and p oduc i i y in he second d ough pe iod
A e age daily empe a u es du ing he expe imen al pe iod in Sep embe anged
be ween 10°C and 20°C (Fig. 2b). The maximum empe a u e was excep ionally high wi h
25.5° C on he 6 h day o he expe imen in Sep embe and lowes on he 11 h day o he
expe imen wi h 12.3° C. The wi hholding o wa e was e lec ed in he ela i e wa e con en
o he lea es, which was educed o a ound 65% bu did no e eal any di e ences be ween
single and ecu en ly d ied plan s (D2 s. C2: P=0.38, Fig. 4a).
C2
Rela i e lea wa e con en [%]
02040
60 80
D2
(a)
Li ing biomass [%]
0 10203040506070
C2 D2
(b)
C2
Rela i e lea wa e con en [%]
02040
60 80
D2
(a)
Li ing biomass [%]
0 10203040506070
C2 D2
(b)
Fig. 4 Lea wa e s a us a he end o he second d ough pe iod 13 days a e wa e had been wi hheld (a),
and pe cen age o li ing biomass o he second ha es , h ee weeks a e he end o he d ough
ea men (b). Means +/- 1 SE a e shown, as e isk indica es signi icance o di e ence (*, P≤ 0.05).
Plan s in single and ecu en d ough ea men s p oduced an almos equal amoun o o e all
(li ing and dead) abo eg ound biomass (1.47g in ecu en d ough ea men (D2) and 1.49g
in single d ough ea men (C2)). Howe e , he pe cen age o li ing biomass, was inc eased
signi ican ly by 7% in plan s subjec ed o ecu en d ough (D2) compa ed o plan s
expe iencing hei i s d ough (C2) (P=0.048) (Fig. 4b). To al li ing biomass was inc eased

Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
115
by 10% in ecu en d ough ea men , al hough his e ec did no p o e o be signi ican
(0.91g in ecu en d ough ea men and 0.82g in single d ough ea men , P=0.18).
3.2.2. Pho osyn he ic pa ame e s
Wi h p og essi e d ough s ess, F /Fm in he single and ecu en d ough ea men
declined, eaching a minimum o he double-s essed plan s (D2) on he 14 h day o he
expe imen , wo days be o e ewa e ing (Fig. 5a). Single-s essed plan s (C2) al eady eached
minimal quan um e iciency on he 11 h day o he d ough and F /Fm alues ose again a e
ha . The loss o lea wa e and pho ochemical e iciency unde ex eme d ough was e lec ed
in a decline o ne pho osyn hesis by mo e han 60% compa ed o ne pho osyn hesis be o e
he d ough ea men s a ed (D2 and C2) (Fig. 5b).
Fig. 5 Cou se o maximum quan um e iciency F /Fm,
measu ed p edawn (a) and ne pho osyn hesis,
measu ed du ing midday (b) in A. ela ius subjec ed o
ecu en o single d ough be o e and du ing he
d ough phase and ewa e ing in Sep embe . Da k g ey
dashed line indica es he s a o he d ough , black
dashed line indica es he end o he d ough and he
s a o ewa e ing in Sep embe . Means +/- 1 SE a e
shown, as e isk indica es signi icance o di e ence
be ween single and ecu en d ough ea men s on
single days (*, P≤ 0.05).
G asses unde ecu en d ough (D2) showed lowe maximum quan um e iciency
compa ed o plan s exposed o a single d ough (C2), om he ele en h day o he expe imen
un il he end o measu emen s, 10 days a e he onse o ewa e ing (Fig. 5a). This educ ion
was signi ican on he las day o measu emen s unde he d ough (14 h day o he
Rewa e ing
345678
Pn [µmolCO
2
m
-2
s
-1
]
Day o expe imen
1 4 7 10 13 16 19 22 25
0.4 0.5 0.6 0.7 0.8
F /Fm
C2- single
D2- ecu en
D ough
(a)
(b)
Rewa e ing
345678
Pn [µmolCO
2
m
-2
s
-1
]
Day o expe imen
1 4 7 10 13 16 19 22 25
0.4 0.5 0.6 0.7 0.8
F /Fm
C2- single
D2- ecu en
D ough
(a)
(b)
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
116
expe imen , P=0.05) and on he i s day a e ewe ing (17 h day o he expe imen , P=0.02)
(Fig. 5a). Du ing d ough and in he pos -d ough eco e y phase, plan s subjec ed o single
(C2) and ecu en d ough (D2) did nei he di e signi ican ly ega ding ne pho osyn hesis,
no anspi a ion ( anspi a ion da a no shown). Howe e , on he 14 h day o he expe imen ,
ne pho osyn hesis o g asses subjec ed o ecu en d ough (D2) was 25% lowe compa ed o
g asses being subjec ed o hei i s d ough (C2), bu his e ec was no signi ican (P=0.11).
A close look a luo escence pa ame e s on bo h o hese days wi h signi ican educ ions in
F /Fm (14 h and 17 h day o expe imen ) e ealed ha he dec ease o F /Fm in plan s subjec ed
o ecu en d ough can be explained by a dec ease in Fm a he han by an inc ease in F0
(Table 2). Two days be o e ewa e ing, Fm was educed by a ound 20% (14 h day o he
expe imen , P=0.07). F0 was non-signi ican ly educed by a ound 6% (P=0.57). Fm was
educed by a ound 10% on he i s day o measu emen s a e ewe ing (17 h day o he
expe imen , P=0.3) as opposed o F0, which was educed by only 0.5% in plan s subjec ed o
ecu en d ough (P=0.94) (Table 2).
Table 2 Maximum luo escence (Fm) and s eady s a e luo escence (F0) wo days be o e ewa e ing (14)
and one day a e ewa e ing (17) in he ecu en and single d ough ea men du ing he expe imen al
pe iod in Sep embe . Means +/- 1 SE a e shown (n=5).
day o expe imen
14 17
Fm
single 0.696 ± 0.0031 0.793 ± 0.0026
ecu en
0.553 ± 0.0030 0.718 ± 0.0025
F0
single 0.157 ± 0.0001 0.170 ± 0.0001
ecu en
0.148 ± 0.0001 0.169 ± 9.9e-5
F /Fm o bo h ea men s eco e ed g adually a e he d ough ea men ended, eaching p e-
d ough alues en days a e ewa e ing had s a ed, on he 26 h day o he expe imen (Fig.
5a). One week a e ewe ing, ne pho osyn hesis had been almos comple ely es o ed,
showing educ ions o only 9% compa ed o p e-d ough alues.
4. Discussion
This s udy in es iga ed, whe he A. ela ius plan s o six mid- and eas e n Eu opean
p o enances can emembe d ough s ess o e an en i e ege a ion pe iod e en a e a
ha es . We hypo hesized ha plan s would no show di e en pe o mance unde ecu en
d ough . This hypo hesis was no con i med, as g asses esponded consis en ly di e en in
ecu en d ough as compa ed o a single d ough , indica ing enhanced pho op o ec ion.
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
117
Su p isingly, his e ec pe sis ed a e o al abo eg ound biomass ha es and eg ow h. The
obse ed changes in eac ion o ecu en d ough a e no in acco dance wi h indings
indica ing educed esis ance o esilience a e ha ing al eady been exposed o d ough s ess
be o e (Llo e e al., 2004; Za alloni e al., 2008 ). Howe e , hese we e conduc ed unde ield
condi ions and Za alloni e al. (2008) in es iga ed communi y esponses. Thus, di e en
ou come o he expe imen s a e no su p ising. The indings a e in acco dance wi h epo ed
s ess imp in e ec s o s ess memo y (B uce e al., 2007). Howe e , o ou knowledge, no
s udy has al eady p o ided e idence ha g asses do emembe d ough s ess e en a e a
ha es and can exhibi imp o ed pe o mance in he ace o epea ed abio ic s ess o e such
a long du a ion. S ess imp in and acclima ion we e p e iously mos ly epo ed o las o
“se e al days” (B uce e al., 2007).
Unde se e e d ough , g asses expe iencing ecu en d ough showed educed
maximum quan um e iciency F /Fm (Fig. 5a). This was mainly ela ed o educ ions in
maximum luo escence, indica ing enhanced dissipa ion o ligh ene gy o p e en
pho odamage (Maxwell and Johnson, 2000). Osmond e al. (Osmond e al., 1993) and A aus
e al. (A aus e al., 1998) sugges ha a co ela ion o educed F /Fm wi h an inc ease o F0
can be in e p e ed as ch onic pho oinhibi o y damage due o he deg ada ion o he D1 p o ein
in he eac ion cen e s. By con as , a cons an F0 and dec easing Fm alues, as in ou s udy,
poin owa ds pho oinhibi ion ela ed o enhanced non-pho ochemical quenching ia he
Xan hophyll cycle and hus indica e pho op o ec ion (A aus e al., 1998). Ano he possible
explana ion o dec eased F /Fm caused by educed Fm alues migh be a educ ion o
chlo ophyll. Un o una ely, we did no measu e chlo ophyll con en in ou s udy. Howe e , a
educ ion o chlo ophyll can be conside ed as a ea u e o acclima ion, as i educes he
possibili y o pho odamage because o an excess o ene gy (Munne-Bosch and Aleg e, 2000).
Ne pho osyn hesis did no e eal any di e ences be ween g asses unde ecu en (D2) and
single d ough (C2), bu showed a end owa ds lowe pho osyn hesis in plan s ecei ing
ecu en s ess unde ex eme d ough (Fig. 5b). This is in acco dance wi h he educed
pho ochemical e iciency in plan s ecu en ly expe iencing d ough (D2).
The esul s o he abo eg ound biomass suppo he hypo hesis o enhanced
pho op o ec ion o double-s essed plan s, as he pe cen age o li ing biomass was
signi ican ly inc eased in plan s expe iencing hei second d ough , al hough o al
abo eg ound biomass o o al li ing biomass we e no signi ican ly al e ed.
Plan s can adap o d ough by enhancing oo g ow h. Howe e , ela i e wa e con en
o he lea es was no signi ican ly enhanced in plan s expe iencing ecu en d ough ,
Manusc ip 4: Do plan s emembe d ough ? Hin s owa ds a d ough memo y in g asses
118
indica ing ha he obse ed esul s can no be explained by changes in oo biomass o
imp o ed wa e up ake mechanisms.
Ecophysiological measu emen s did no e eal any consis en di e ences be ween
ecu ing (D2) and single d ough (C2) in he pos d ough eco e y phase. Maximum
quan um e iciency wo days a e ewe ing in g asses subjec ed o ecu ing d ough was
signi ican ly lowe compa ed o g asses subjec ed o hei i s d ough . This was mo e likely
ela ed o inc eased s ess le els unde d ough a he han a lowe eco e y a e. Some s udies
indica ed ha eco e y a e depends on expe ienced s ess le el (Miyashi a e al., 2005; Resco
e al., 2009). The signi ican ly lowe pe cen age o dead biomass in plan s subjec ed o
ecu ing d ough can be a sign o quicke eco e y, bu is mo e likely o be a sign o
imp o ed pho op o ec ion, as discussed abo e. In acco dance o o he s udies, he eco e y o
ecophysiological pa ame e s was qui e as , almos eaching p e-s ess le els a e abou one
week (Galle e al., 2007; Galmes e al., 2007).
We did no elucida e unde lying molecula o biochemical mechanisms o
acclima ion in his s udy, as we we e in e es ed in he e ec s o ecu en d ough on
ag icul u al ele an pe o mance pa ame e s. Thus, we can only hypo hesize abou po en ial
long-las ing acclima ion p ocesses in he g asses. The obse ed pheno ypic plas ici y could be
ei he explained by belowg ound dynamics o by long-las ing changes in gene exp ession,
ende ing he plan s mo e pe missi e o eac quickly o ecu en s ess, e.g. epigene ic
p ocesses (Aubin-Ho h and Renn, 2009;Molinie e al., 2006; Bi d, 2007; Bossdo e al.,
2008). Ve hoe en e al. (Ve hoe en e al., 2010) ecen ly showed ha s ess induces changes
in me hyla ion pa e ns and ha hese pa e ns a e he i able. An in es iga ion o he changes
in me hyla ion pa e ns as a esponse o d ough and a link o obse ed me hyla ion pa e ns o
s ess esponse a e e y p omising. Fu he mo e, we could only in es iga e six p o enances o
A. ela ius plan s, which o igina ed mainly om a eas in Eu ope wi h qui e simila clima ic
condi ions. O he , mo e di e en p o enances we e no su i ing in su icien eplica es o
ou s udy. Ne e heless, an ex ension o ou expe imen o o he p o enances and plan
g oups seems p omising, as hey may e eal di e en acclima ion pa e ns and he e o e also
di e en esponses o ecu en d ough .
5. Conclusion
To conclude, ou s udy indica es ha g asses unde d ough e ain a long-las ing s ess
imp in ha acili a es a as e and mo e p o ec i e esponse owa ds a ecu en d ough .
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
125
He e, eigh p o enances o P. nig a om au och honous o igins and om sou he n
Ge many we e es ed o hei cold ha diness in a common ga den expe imen in sou he n
Ge many. We hypo hesized ha (1) cold ha diness di e s be ween p o enances, wi h
p o enances om colde o igins displaying supe io cold ha diness, and ha (2) cold
ha diness is a ec ed by clima ic expe iences o he indi iduals wi h d ough inc easing cold
ha diness and wa ming dec easing cold ha diness. We u he expec ed ha (3) di e ences in
cold ha diness be ween p o enances a e physiologically- ela ed o he con en o soluble
ca bohyd a es and lipid composi ion o he needles, and ha (4) he (sub-) medi e anean
species P. nig a is less os - ole an han ee species na i e o Cen al Eu ope, while (5) cold
ha diness o he ine oo s o P. nig a is high compa ed o cold ha diness o i s oliage as i
na u ally occu s in egions wi hou con inuous snow co e .
2. Ma e ial and me hods
Ju eniles o P. nig a om eigh p o enances h oughou Eu ope we e exposed o
di e en clima e change scena ios (wa ming and ex eme d ough ) in a common ga den
expe imen . Cold ha diness was de e mined by he Rela i e Elec oly e Leakage me hod
(REL) in wo consecu i e win e s. The expe imen was es ablished in Bay eu h, Ge many
(49°55’19” N, 11°34’55” E) in Ma ch 2009. The long- e m mean annual empe a u e o he
si e is 8.2°C, whe eas long- e m mean annual p ecipi a ion is 724 mm.
2.1. Expe imen al design
Eigh p o enances o P. nig a (Figu e 1; Table 1) we e ob ained as seeds and cul i a ed
a he Ba a ian Ins i u e o Fo es Seeding and Plan ing (ASP) in Teisendo , Ge many om
Ap il 2008 o Ap il 2009.

Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
126
Figu e 1: O igins o he a ge p o enances (open ci cles) wi hin he dis ibu ion o P. nig a (black lines
and do s o agmen ed popula ions Isaje e al., 2004). X indica es he expe imen al si e. Abb e ia ions
o p o enances a e speci ied in Table 1. G ey scales display he mean minimum empe a u e o he
pe iod 1950 o 2000 wi h a 5’ spa ial esolu ion (Hijmans e al., 2005).
These p o enances a e pa o an in e na ional long- e m p o enance ial which s a ed
in 2009 (Hube , 2011). The p o enances s em om au och honous popula ions o P. nig a
excep o he p o enance om Zellingen, Ge many, which was in oduced om Aus ian
sou ces in 1909. Subspecies iden i ies o he p o enances a e assigned geog aphically and
mo phologically (Table 1), as gene ic analyses a e no ye a ailable (Hube , 2011).
Table 1 O igins o a ge p o enances used in he expe imen wi h co esponding clima ic in o ma ion.
Skie: Iden i ica ion numbe in an in e na ional p o enance ial (Hube , 2011). MAT: Mean Annual
Tempe a u e; MinT: Mean Minimum Tempe a u e; MAP: Mean Annual P ecipi a ion; P ecip.
Seasonali y: Coe icien o a ia ion in mean mon hly p ecipi a ion sum. All clima e da a o he pe iod
1950 o 2000 om wo ldclim (Hijmans e al., 2005).
P o enance Coun y Subspecies Skie No h Eas Al i ude
(m)
MAT
(°C)
MinT
(°C)
MAP
(mm)
P ecip.
Seasonali y
DE Ge many
nig a 01 49°53'17" 09°43'16" 290 9.2 -3.1 587 18
AU Aus ia
nig a 07 47°46'00" 16°11'00" 369 8.4 -4.9 712 33
YU Se bia
nig a 12 43°49'39" 19°35'22" 866 8.7 -5.6 964 17
HR C oa ia
nig a/
dalma ica
14 43°26'00" 17°13'00" 256 13.2 1.2 1108 33
IT.N I aly
nig a 17 45°42'00" 13°49'00" 372 11.4 -1.2 1212 17
IT.S I aly
la icio 19 39°18'08" 16°20'22" 1500 9.0 2.2 1300 48
FR1 F ance
nig a 23 44°09'10" 05°52'30" 549 10.7 -2.9 789 16
FR2 F ance
la icio 24 44°24'18" 03°58'39" 581 10.8 -0.9 745 19
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
127
The C oa ian p o enance s ems om a loca ion e y close o one o he ew
au och honous s ands o P. nig a subspecies dalma ica and i s assignmen o he subspecies
nig a is somewha ques ionable. The seedlings we e anspo ed o Bay eu h and indi idually
plan ed in o 4-li e plas ic po s illed wi h sandy sil (pH 7.3, o al C 1.9%, o al N 0.15%,
plan a ailable NO3--N 22.5 mg l-1; plan a ailable NH4+-N 1.8 mg l-1). Selec ion o he plan s
occu ed andomly o each p o enance om all hose plan s ali e a he plan ing da e. The
mean plan size a he s a o he expe imen was 12.2 cm ± 2.5 cm SD.
The po ed indi iduals we e exposed o he ully c ossed h ee old ac o ial combina ion
o (1) a d ough manipula ion (d ough and con ol) and (2) a con inuous wa ming
manipula ion (wa ming and e e ence) and (3) he p o enance ea men (eigh p o enances).
The wo clima e ea men s we e c ossed esul ing in ou clima e manipula ions (con ol,
d ough , wa ming, wa ming & d ough ), ha we e eplica ed h ee imes, esul ing in 12
expe imen al uni s in o al. The p o enance ea men was nes ed wi hin each expe imen al
uni . Each p o enance was u he eplica ed wi h se en plan s pe expe imen al uni (nes ed
eplica es), esul ing in 21 plan s pe ac o ial combina ion o he h ee- ac o ial design and
672 plan s o e all. Each expe imen al uni was co e ed by a single ain-ou shel e (11 m by 7
m, 3.8 m high) cons uc ed o a s eel ame (GlasMe all Rieme GmbH) and co e ed wi h a
anspa en polye hylene shee (0.2 mm, SPR5, He mann Meye GmbH) enabling an almos
90% pene a ion o pho osyn he ically-ac i e adia ion. The edge o he ain-ou shel e s was
a a heigh o 80 cm.
The con ol i iga ion egime simula ed he local daily 30-yea a e age p ecipi a ion.
The applica ion was ca ied ou wice a week wi h collec ed ain wa e . The d ough ea men
consis ed o 42 days wi hou p ecipi a ion, which ep esen s he local s a is ical 1000-yea
ecu ence d ough e en . D ough du a ion was no a p io i se be o e he manipula ions. We
moni o ed plan pe o mance du ing he ea men and would ha e s opped he ea men
when ei he 66% o he plan s showed wa e s ess symp oms (discolo a ion o oliage) o
when 33% o he plan s exhibi ed le hal s ess, o when he local 1000-yea ex eme would be
eached. The la e condi ion was se because we assume ha e en s wi h mo e han 1000 yea
ecu ence ime a e no oo ealis ic e en when changing equencies o ex emes due o
clima e change a e acknowledged (Schä e al., 2004). The same p o ocol was applied in a
pa allel expe imen wi h ou g ass species (Beie kuhnlein e al., 2011), which all showed
se e e d ough symp oms a e abou 20 days, emphasizing he high d ough ole ance o
Pinus nig a. The d ough ea men s a ed on May 27 h 2009 and esul ed in he soil mois u e
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
128
alling below he pe manen wil ing poin (pF = 4.2) o he soil app oxima ely h ee weeks
a e he s a o he ea men (Figu e 2). In he e-we ing phase each indi idual in he
d ough ea men s ecei ed 240, 280 and 300 ml on h ee days wi hin one week (in o al 820
ml o 36 mm). Following ha , he po s we e i iga ed acco ding o he con ol p ecipi a ion
ea men . To al amoun o p ecipi a ion in he d ough ea men was 13% lowe han in he
con ol o e he yea . The d ough was simula ed in he i s yea o he expe imen only.
Th oughou he second yea , all plan s ecei ed con ol i iga ion.
Figu e 2: Tempe a u e a mean plan heigh , snow co e and soil mois u e (-2.5 o -7.5 cm) o e he
cou se o he expe imen . Sampling da es a e indica ed by a ows.
The wa ming ea men was pe o med con inuously un il Oc obe in he i s yea o
he expe imen and om Ap il o he end o he expe imen in Janua y o he second yea . The
wa ming manipula ion ook place bo h passi ely (wind-shel e s which educed he wind speed
by 70 % and black loo -co e s e sus whi e loo co e s) and ac i ely (IR- adia ion wi h
app oxima ely 30 W pe m²), which inc eased he ai empe a u e a plan heigh by 1.6°C on
a e age when he wa ming ea men was a ec ed (Figu e 2). Maximum di e ences we e
5.2°C (single measu emen s) o 3.6°C o daily mean empe a u e. The ou h ea men was a
combina ion o d ough and wa ming. The wa ming inc eased he d ough e ec , educing he
soil mois u e by ano he 1.5% on a e age (Figu e 2).
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
129
Du ing he i s win e o he expe imen , plan s we e kep ou side he shel e s in a sand
bed om Oc obe o Ap il. Figu e 2 illus a es ha he plan s we e co e ed by snow du ing
he coldes pa s o he i s win e . Fo he second win e , plan s we e kep inside he shel e s
wi h he wa ming ea men ongoing.
2.2. Response pa ame e s
Cold ha diness was quan i ied by a sligh ly modi ied e sion o he ela i e elec oly e
leakage me hod (REL) o ex-si u samples acco ding o S imbeck e al. (2007): P e- es s
e ealed no di e ences in absolu e alues when he samples we e ozen wi h o wi hou 1ml
solu ion con aining an ice nuclea o , p esumably because he su ace o he samples was we
and oze a a ound 0°C anyhow. Fu he mo e, highe eezing a es we e applied. A a a e o
0.6°C/h (S imbeck e al. 2007) i would ha e aken 3.5 days o each ou minimum
empe a u e, whe eas commonly he a e o 6°C/h is applied (e.g. Su inen 1992, Schabe g
2008). Two needles om he cu en yea we e sampled pe indi idual in mid-win e o bo h
yea s (Janua y 20 h in 2010 and Janua y 31s in 2011), insed wi h de-ionized wa e , and cu o
0.5 cm. Samples om he se en nes ed eplica es pe p o enance and expe imen al uni we e
combined o o m one mixed sample, homogenized and subsequen ly di ided in o se en
subsamples subjec ed o di e en empe a u e le els o one hou (+4.5°C, -7.5°C, -14.5°C, -
23°C, -33°C; -40°C, -196°C (liquid N)) using a con olled en i onmen chambe (Lich -
The mos a e Typ 1301, RUMED) and a manually con olled chain o eeze s sequen ially a
he lowes empe a u es. Ini ial elec oly e leakage was de e mined in 16 ml 0.1% / T i on
X-100_Bides a e 24 h and he inal elec oly e leakage was de e mined a e au ocla a ion
o he samples. Elec oly e leakage was quan i ied by he conduc i i y o he solu ion a 25°C
measu ed wi h a WTW inolab pH/Cond 720. Cold ha diness is exp essed as he LT50 o
each mixed sample, es ima ed by non-linea eg ession o he REL e sus he empe a u e
le els using he o mula by Ande son e al. (1988):
(1)
YT is he REL a empe a u e T, Ymin is he asymp o ic alue o he esponse a iable in
uninju ed issue, Ymax is he asymp o ic alue a maximum low- empe a u e s ess, k
ep esen s he s eepness o he esponse cu e, and Tm is he midpoin o he symme ical
cu e (an es ima e o LT50). Cu e i ing was ca ied ou using a quan ile eg ession and he
unc ion “nl q()” om he so wa e package “quan eg” (Koenke , 2006).
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
130
The mul i ude o di e en echnical p o ocols o REL used in he li e a u e ( eezing
wi h o wi hou addi ional solu ion, a ious eezing a es and du a ions, e c.) limi s he
compa abili y be ween s udies s ongly. Howe e , he ela i e di e ences wi hin a p o ocol
should be obus and mo e o less independen o e.g. eezing a es (Su inen e al 1992).
The e o e, we s ick o he in e p e a ion o ela i e di e ences wi hin ou s udy and minimize
he discussion o absolu e alues.
Cold ha diness o needles om he cu en yea o e minal buds was addi ionally
de e mined o ju enile and adul indi iduals o he mos impo an local ee species (P.
syl es is, Picea abies, Fagus syl a ica and Que cus pe aea). Samples we e aken on 26 h o
Janua y in he i s win e . Th ee mixed samples o se en indi iduals each we e ob ained
om a nea by o es (lowland si e: 350 m asl) and, o he ju enile s age o he wo coni e s,
om a highland si e (Walds ein, Fich elgebi ge, 760 m asl) abou 50 km no hwes o he
expe imen al si e.
In he second win e , ca e ully exca a ed ine oo s o wo p o enances (FR1 and IT.S)
exhibi ing low and high cold ha diness in hei oliage in he i s yea we e analyzed o hei
cold ha diness by applying he same p o ocol as o he needles.
Mean annual minimum empe a u es o he pe iod 1950 o 2000 (mean empe a u e o
he coldes day o he yea s 1950-2000) o each geog aphic o igin o he p o enances we e
e ie ed om wo ldclim (Hijmans e al., 2005) and used as indica o s o minimum
empe a u es (Table 1). We assume ha he ela i e di e ences be ween geog aphic o igins
ha e been sui ably e lec ed, al hough hese alues exceed he absolu e minimum
empe a u es due o daily a e aging ( o ou expe imen al si e he minimum empe a u e
based on wo ldclim is -3.5°C while he absolu e annual minimum empe a u es be ween 1998
and 2011 a an hou ly esolu ion anged be ween -10.8 and -25.5°C). An o dina y leas
squa es eg ession be ween his indica o and cold ha diness was applied o he con ol
ea men in o de o de ec local adap a ion o la e os e en s.
Soluble ca bohyd a es we e quan i ied in he i s win e o wo p o enances exhibi ing
low and high cold ha diness, espec i ely (FR1 and IT.S). Mixed samples o one needle om
he se en plan s pe expe imen al uni we e aken, immedia ely ozen in liquid ni ogen and
s o ed a -30°C. F ozen ma e ial was g ound in a ball mill; soluble ca bohyd a es o 20 mg o
plan ma e ial we e ex ac ed in 50 % me hanol and analyzed using he an h one me hod
(Klebe e al., 1997). Ex inc ion was measu ed a 620 nm. We used known concen a ions o
Glucose as a s anda d.

Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
131
Lipid composi ion was ob ained o he same wo p o enances (FR1 and IT.S).
Epicu icula wax lipids including alkanes as he mos abundan wax componen we e
eco e ed by insing needles o 60 seconds in dichlo ome hane (DCM), which esembles
s anda d echniques using chlo o o m (Radle and Ho n, 1965). A e emo al o he
epicu icula wax lipids, needles we e g ound o a ine powde using a ball mill (Re sch
M200). In e nal waxes we e eco e ed by s anda d Soxhle ex ac ion using a mix u e o
DCM/Me hanol (93:7) (Wiesenbe g e al., 2010). Ex ac s o in e nal and epicu icula waxes
we e d ied and sequen ially sepa a ed using solid-phase ex ac ion in o lipid ac ions
including a y acid and alkane ac ions (Wiesenbe g e al., 2010). The ollowing sec ion only
discusses he esul s o he alkane ac ion as a ep esen a i e componen o epicu icula wax
lipids and he a y acids o in e nal lipids as he dominan compound class o cell memb anes.
O he ac ions we e also analyzed, bu no signi ican di e ences in hei dis ibu ion pa e ns
we e ob ained. Aliquo s o deu e ia ed s anda ds (D39C20 acids and D50C24 alkane,
espec i ely) we e added o he lipid ac ions o compound iden i ica ion and quan i ica ion.
Fa y acid ac ions we e de i a ized using BSTFA (N,O- Bis ( ime hylsilyl)
i luo oace amide) o 1h a 80°C, whe eas alkanes did no equi e any u he p epa a ion.
All ac ions we e measu ed using gas ch oma og aphy coupled wi h lame ioniza ion
de ec ion (Agilen 7890).
In addi ion o lipid dis ibu ion pa e ns, molecula p oxies we e also de e mined o
e alua e he di e ences be ween p o enances and clima e manipula ions. The a e age chain
leng h (ACL) o lipids is in luenced by lipid biosyn hesis and egula es he wa e epellency
o hyd ophobic hyd oca bon chains o a y acids in cell memb anes as well as a y acids and
alkanes in epicu icula waxes (Kola ukudy e al., 1976). Ini ially, he ACL was used o
di e en ia e plan and mic obial sou ces o o ganic ma e in e es ial sedimen s (B ay and
E ans, 1961):
ACL = Σ (zn * n) / Σ (zn) (2)
whe e n is he numbe o ca bons and zn he amoun o a y acids o alkanes wi h n
ca bons. Ano he pa ame e o ob ain he di e ences in he lipid biosyn hesis as a ec ed by
en i onmen al s ess is he p edominance o odd e sus e en alkanes, he so-called ca bon
p e e ence index (CPI: Kola ukudy e al., 1976):
CPI = [(Σ n-C25-33 odd / Σ n-C24-32 e en) + (Σ n-C25-33 odd / Σ n-C26-34 e en)]/2 (3)
The deg ada ion o alkanes and a less e ec i e syn hesis o he p edominan odd alkanes in
waxes lead o a educ ion o he CPI unde en i onmen al s ess (Wiesenbe g e al., 2008).
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
132
An analysis o a iance (ANOVA) combined wi h linea mixed e ec models we e
applied o es o he main and in e ac i e e ec s o he h ee ac o s: p o enance, d ough ,
and wa ming on cold ha diness (LT50), soluble ca bohyd a e con en and lipid composi ion.
Including he expe imen al uni as a andom ac o accoun ed o he spli -plo design
(Pinhei o and Ba es, 2004). Da a we e log ans o med o imp o e he homogenei y o
a iances and he no mali y o esiduals i necessa y. All s a is ical analyses we e conduc ed
wi h he so wa e R 2.11.1 (R De elopmen Co e Team, 2010) and he addi ional packages
“nlme” and “quan eg”.
3. Resul s
3.1. Local adap a ion in cold ha diness
Mean cold ha diness di e ed be ween he p o enances by abou 10°C in bo h win e s
(Figu e 3).
Figu e 3: Cold ha diness (LT50) as a ec ed by geog aphic o igin (p o enances; le ) and p eceding
clima ic condi ions (d ough and wa ming in in e ac ion; igh ) in he i s (uppe panel) and second
(lowe panel) yea o he expe imen . ANOVA- esul s a e p o ided pe yea wi h signi ican e ec s in
bold. Mean alues and s anda d e o s a e shown o 84 indi iduals pe ba o he p o enances and 168
indi iduals pe ba o he clima e ea men s. No e ha he d ough manipula ion only ook place in he
i s summe o he expe imen . The wa ming ea men s opped h ee mon hs be o e sampling in he i s
yea while unning h oughou sampling in he second yea .
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
133
The LT50 alues anged be ween a minimum o -21.2°C o p o enance FR2 and a maximum
o -32.1°C o p o enance FR1 in he i s win e ; and be ween a minimum o -23.2°C o
p o enance FR2 and a maximum o -33.1°C o he p o enance om Se bia (YU) in he
second win e . P o enances om colde o igins gene ally displayed supe io cold ha diness
(Figu e 4). Signi ican co ela ions ( ² = 0.77 in he i s win e and ² = 0.80 in he second
win e ) be ween cold ha diness and mean minimum win e empe a u e a he o igins we e
ound o he au och honous p o enances in bo h yea s i he p o enance om C oa ia was
excluded om he analyses. When included, no signi ican co ela ion was ound in he i s
win e , while he co ela ion was weake ( ² = 0.59) while s ill emaining signi ican in he
second win e .
Figu e 4: Local adap a ion in cold ha diness depending on he mean minimum empe a u e a he o igin.
Linea eg essions a e shown o all au och honous p o enances (DE, open iangle, no included)
excluding he p o enance om C oa ia (HR, open ci cle), as i s au och honous s a us is ques ionable.
Resul s o he eg ession including he p o enance om C oa ia a e gi en in pa en heses. Cold ha diness
(LT50) displays he mean o he con ol ea men pe p o enance (n = 21).
3.2. Clima ic expe iences al e cold ha diness
Cold ha diness was a ec ed by he clima ic expe iences o he indi iduals. The ex eme
summe d ough inc eased cold ha diness by 3.9°C on a e age in he i s win e and he e
was a non-signi ican end in he same di ec ion in he second win e a e he d ough
manipula ion (Figu e 3). Unexpec edly, he summe wa ming om he i s yea esul ed in
inc eased cold ha diness o 3.4°C on a e age while he yea - ound wa ming o he second
yea esul ed in no signi ican e ec , al hough he end ollowed he same di ec ion as in he
i s yea . In e es ingly, he d ough and he wa ming e ec in he i s yea we e no addi i e
(ANOVA, in e ac ion be ween d ough and wa ming: F = 16.0; p = 0.004), esul ing in lowe
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
134
cold ha diness in he un ea ed a ian and compa able, high cold ha diness in he o he h ee
clima e manipula ions (Figu e 3).
3.3. Physiological easons o a ying cold ha diness
The amoun o soluble ca bohyd a es in he needles inc eased by 25.9% in a p o enance
exhibi ing high cold ha diness compa ed o a p o enance showing low cold ha diness (Table
2; ANOVA: F = 15.3; p = 0.004). The d ough manipula ion had no signi ican e ec on he
ca bohyd a e concen a ion (F = 0.0; p = 0.889).
Table 2: Compa ison o ca bohyd a e con en and a e age chain leng h (ACL) o a y acids o cu en
yea needles be ween wo p o enances exhibi ing low (IT.S) and high (FR1) cold ha diness. Samples aken
in he i s win e o he expe imen , mean ± s anda d de ia ion p o ided, n = 3.
Cold ha diness o sou ce Con ol D ough
Cold ha diness (LT50 in °C) high -25.8 ± 2.3 -36.7 ± 1.0
low -17.5 ± 1.6 -26.8 ± 4.5
Soluble ca bohyd a es (‰TM) high 118.4 ± 9.4 111.9 ± 9.5
low 89.0 ± 15.9 93.7 ± 3.0
ACL o epicu icula wax a y acids high 18.8 ± 0.2 17.7 ± 0.2
low 18.1 ± 0.0 17.8 ± 0.1
ACL o in e nal a y acids high 17.8 ± 0.5 17.6 ± 0.1
low 17.5 ± 0.1 17.1 ± 0.3
ACL o epicu icula wax alkanes high 27.5 ± 0.1 27.6 ± 0.0
low 27.5 ± 0.1 27.5 ± 0.1
CPI o epicu icula wax alkanes high 9.3 ± 0.2 10.4 ± 0.2
low 12.0 ± 0.6 11.9 ± 0.4
The composi ion o in e nal a y acids (ACL) as main componen s o cell memb anes
did no di e signi ican ly be ween a p o enance exhibi ing high cold ha diness and a
p o enance showing low cold ha diness (F = 3.7; p = 0.092). Likewise, no e ec o he
d ough manipula ion was ound (F = 1.7; p = 0.222). Fo he epicu icula wax lipids, he
p o enance wi h he high cold ha diness was cha ac e ized by a sligh ly highe ACL (3.9 %; F
= 5.2; p = 0.051) han ha wi h he low cold ha diness. The d ough ea men led o a gene al
dec ease in ACL (F = 22.0; p = 0.002), which was s onge o he plan s wi h a high (5.5 %)
a he han a low cold ha diness (1.5 %; in e ac ion be ween p o enance and d ough
manipula ion: F = 7.7; p = 0.024).
Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
141
selec ion o os - ole an p o enances could be based on he cu en clima ic condi ions
wi hin he species anges. Howe e , ou inding ha clima ic expe iences wi hin he li e o
single plan s al e cold ha diness indica es ha p o enance ials unde con ol condi ions may
be misleading unde changing clima ic mean and ex eme condi ions. The mul i ude o
possible clima ic a iables o be selec ed o and unce ain ies conce ning u u e clima es
imply ha he sea ch o bes -adap ed p o enances should no be he only s a egy. In
addi ion, managemen ac ions which p omo e gene ic di e si y (e.g. suppo ing na u al
egene a ion and addi ion o gene ically di e se ma e ial) a e c ucial as gene ic di e si y
enables o ganisms o con inue adap ing and e ol ing o new condi ions wi hin one o se e al
gene a ion cycles (Hosius e al., 2006; Schabe g e al., 2008b). Fu he mo e, he ole o
he bi o es and diseases unde changing clima e equi es de ailed in es iga ions. Fo ins ance,
a needle bligh known as he “ ed band disease” (Do his oma sep ospo a) is epo ed o
inc ease in impo ance o e ecen yea s in P. nig a (Isaje e al., 2004), a de elopmen ha
may be ela ed o clima e change (Wa e al., 2011).
Ul ima ely, ee species esponses should be ega ded in he con ex o popula ions
unde compe i i e p essu e. The ad an age o common ga den expe imen s is ha hey can
de ec he spec um o possible species-speci ic esponses. Ne e heless, he e is a need o es
he ob ained esul s in communi ies whe e he compe i i e balance migh ampli y o bu e
esponses.
5. Conclusions
Cold ha diness o Pinus nig a oliage is highly a iable be ween p o enances and
shows signs o local adap a ion o p e ailing minimum empe a u es a he o igin. Bo h se e e
d ough e en s and summe wa ming can inc ease cold ha diness, indica ing ha he
in e ac ion o di e en clima e pa ame e s leads o unexpec ed esul s and ha win e su i al
can be al e ed by clima ic e en s du ing he g owing season. Physiologically, cold ha diness
is ela ed o soluble ca bohyd a e con en and lipid composi ion. In e es ingly, a ia ion o
cold ha diness o he needles wi hin he (sub-) medi e anean species P. nig a was highe han
be ween his species and o he species common o he empe a e zone o Cen al Eu ope.
Taken oge he , ou esul s imply ha he cold ha diness o he oliage o P. nig a is adap i e
o long- e m g owing condi ions a he o igin (gene ic he i age) and o sho - e m al e a ions
o hese condi ions (indi idual plas ici y), while i s hin s sugges ha cold ha diness o he

Manusc ip 5: Cold ha diness o Pinus nig a A nold as in luenced by geog aphic o igin, wa ming, and
ex eme summe d ough
142
oo s is high and p obably no unde selec i e p essu e cu en ly. Ou da a om mid-win e
sugges s ha below- and abo e-g ound cold ha diness o selec ed p o enances appea o be
well adap ed o cul i a ion in empe a e egions as an adap a ion s a egy agains he ad e se
e ec s o clima e change in d y habi a s. Howe e , wi h espec o la e sp ing and ea ly
au umn os e en s, he empo al pa e n o os ha diness wi h po en ial in a-speci ic
di e ences should be in es iga ed in mo e de ail. Be o e ansloca ions a e ecommended,
u he in es iga ions a e equi ed, e.g. explo ing he ole o bio ic in e ac ions unde
changing clima ic condi ions. Gene ally, wi hin-species di e si y should be conse ed a he
species le el and imp o ed in an h opogenically ounded s ands in o de o allow o adap ion
o clima e change.
6. Acknowledgemen s: This s udy was unded by he Obe ankens i ung (OF a_02631) in
coope a ion wi h he "Ba a ian Clima e P og amme 2020" in he join esea ch cen e
“FORKAST” and he Ba a ian S a e Minis y o he En i onmen and Public Heal h
(ZKL01Ab 7_18456). We hank Ch is ian Schemm, Elke König, S e an König, Ch is ine Pilsl
and nume ous s uden wo ke s and in e ns o hei ou s anding help du ing he ield wo k.
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Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency
145
Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age
quali y o empe a e g assland la gely independen o mowing equency
Ag icul u e, Ecosys ems and En i onmen , 2012, 148: 1-10.
Julia Wal e a*, Ke s in G an b∗, Ca l Beie kuhnleinc, Jü gen K eylingc, Michael Webe d, Anke
Jen schb
aConse a ion Biology, Helmhol z Cen e o En i onmen al Resea ch- UFZ, Pe mose s aße 15, 04318 Leipzig,
Ge many, Julia.wal e @u z.de, phone:+49-341-2351654, ax: :+49-341-2351470, co esponding au ho
bDis u bance Ecology, Bay eu h Uni e si y, 95440 Bay eu h, Ge many
cDepa men o Biogeog aphy, Bay eu h Uni e si y, 95440 Bay eu h, Ge many
dDepa men o Plan Physiology, Bay eu h Uni e si y, 95440 Bay eu h, Ge many
Highligh s
1. G assland was subjec ed o inc eased ain all a iabili y and mowing equency.
2. Inc eased ain all a iabili y educes g assland p oduc i i y and o age quali y.
3. Mo e equen mowing ini ially inc eases and la e on dec eases p oduc i i y.
4. Mowing egime does mos ly no in e ac wi h ain all a iabili y manipula ions.
5. Su icien o e all ain all amoun is impo an o g assland esilience.
Abs ac
Clima e models indica e ha global wa ming will s imula e a mosphe ic exchange
p ocesses and inc ease ain all a iabili y, leading o longe d y pe iods and mo e in ense
ain all e en s. Recen s udies sugges ha bo h he magni ude o he ain all e en s and hei
equency may be as impo an o empe a e g assland p oduc i i y as he annual sum.
Howe e , un il now in e ac i e e ec s be ween land managemen p ac ice, such as mowing
equency, and ain all a iabili y on p oduc i i y and o age quali y ha e no been s udied in
de ail. He e, we p esen he da a om a ield expe imen (EVENT II) in which a Cen al-
Eu opean g assland was subjec ed o inc eased sp ing ain all a iabili y (low, in e media e
and ex eme ain all a iabili y wi hou any change o he ain all amoun ) and inc eased
mowing equency ( ou imes compa ed o wice a yea ). We assessed biomass p oduc ion,
∗ au ho s con ibu ed equally o he publica ion
Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency
146
o age quali y pa ame e s, oo -leng h and shoo - oo a io. Enhanced sp ing ain all
a iabili y educed midsumme p oduc i i y and he lea N and p o ein concen a ions o a
a ge species, bu did no exe any long- e m e ec s on biomass p oduc ion and o age
quali y in la e summe . Howe e , he inc eased sp ing ain all a iabili y educed
abo eg ound ne p ima y p oduc i i y by 15 %. Mo e equen mowing inc eased p oduc i i y
in he i s yea o he s udy, bu dec eased p oduc i i y a he end o he second yea , showing
a decline in he po en ial o o e compensa ion a e a his o y o mo e in ense mowing.
Gene ally, mo e equen mowing dec eased he shoo - oo a io and inc eased he
concen a ion o lea N. Inc eased mowing equency nei he bu e ed, no ampli ied he
ad e se e ec s o ain all a iabili y on p oduc i i y, bu made lea N concen a ions in ea ly
summe mo e esponsi e o al e ed ain all pa e ns. These esul s highligh he ac ha e en
ela i ely small and sho - e m al e a ions o ain all dis ibu ion can educe p oduc ion and
o age quali y, wi h li le bu e ing capaci y o al e ed mowing equency. Compa isons wi h
p oduc i i y da a om he i s yea o he s udy, in which bo h, ain all dis ibu ion and
ain all amoun we e modi ied, demons a e he c ucial ole o su icien mois u e (annual
ain all amoun ) o g assland esilience: In his i s yea , nega i e e ec s o ex eme ain all
a iabili y las ed un il he end o he yea . To conclude, inc eased ain all a iabili y unde
clima e change will likely a ec ag icul u al yield in empe a e meadows. Managemen
s a egies o bu e hese e ec s ha e ye o be de eloped.
Keywo ds: EVENT II expe imen , ex eme wea he e en , ainou -shel e , o age quali y,
Alopecu us p a ensis, T i olium p a ense
1. In oduc ion
Clima e change is p ojec ed o modi y no only annual p ecipi a ion sum, bu also o
esul in mo e ex eme ain all egimes in many pa s o he wo ld (IPCC 2007; Jen sch and
Beie kuhnlein, 2008). This will cause mo e se e e d ough pe iods as well as an inc ease in
he equency and magni ude o ex eme p ecipi a ion e en s (T enbe h e al., 2003, Min e
al., 2011). E idence is moun ing ha he equency and se e i y o d ough s and ex eme
p ecipi a ion e en s has al eady inc eased o e ecen decades in many egions (Blenkinsop
and Fowle , 2007; Haylock and Goodess, 2004; IPCC 2007).
P ima y p oduc i i y and ecosys em unc ioning in e es ial ecosys ems a e s ongly
in luenced by he annual amoun o p ecipi a ion (Sala e al., 1988). Howe e , ecen esea ch
sugges s ha ain all a iabili y may exe an e en s onge in luence on ecosys em

Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency
147
unc ioning, whe e especially empe a e g assland sys ems seem o be esponsi e o changes
in ain all a iabili y. In g assland, mo e ex eme ain all egimes (less, bu mo e in ense
ain all e en s) a ec ANPP (abo eg ound ne p ima y p oduc i i y) (Ba e e al., 2002;
Fay, 2009; Heisle -Whi e e al., 2009; Knapp e al., 2002), ca bon cycling (Chou e al., 2008;
Fay, 2009; Ha pe e al., 2005) and N mine aliza ion (Ba e e al., 2002, Heisle -Whi e e al.,
2009). The la e may in u n a ec lea quali y in e ms o N o p o ein con en . La ge
educ ions in ANPP ha e been shown in mesic g assland in esponse o mo e ex eme ain all
pa e ns (Fay e al., 2003; Heisle -Whi e e al., 2009; Knapp e al., 2008).
In addi ion o he ain all amoun and a iabili y, land managemen s a egies, such as
mowing equency, can a ec p oduc i i y and lea li e quali y in managed g assland. Mo e
equen cu ing is known o inc ease lea N con en . Howe e , whe he o no mowing
inc eases o dec eases he p oduc i i y o g assland depends on he mowing in ensi y, e.g.
mowing his o y, mowing equency and cu ing heigh (G een and De ling, 2000;
McNaugh on, 1979; Tu ne e al., 1993; Weigel e al., 2009). Mowing o de olia ion is likely
o al e he esponse o ain all a iabili y by al e ing plan communi y composi ion
(Swemme and Knapp, 2008). Fu he mo e, a educ ion o anspi a i e issue al e s wa e
up ake and consump ion and he e o e eac ion owa ds ain all (Hei schmid e al., 1999;
McNaugh on, 1979; Yang and Midmo e, 2004). Cu en ly, a knowledge gap exis s on how
land managemen p ac ices, such as mowing equency, a e in e ac ing wi h mo e ex eme
ain all egimes: Inc eased mowing equency migh bu e he e ec s o ain all a iabili y
on g assland, diminishing he ampli ude o he esponse owa ds ain all ex emes (Swemme
and Knapp, 2008). A s udy by Be nha d -Röme mann e al. (2011) indica es ha clima e
pa ame e s ge less impo an o biomass p oduc ion unde in e media e mowing equencies.
Howe e , land managemen s a egies migh also ampli y he e ec s o ain all a iabili y. To
ou knowledge, his is he i s s udy o expe imen ally manipula e mowing and ain all
pa e ns in Eu opean managed g assland (meadows) in o de o iden i y any po en ial
in e ac ions be ween ain all a iabili y and mowing equency.
The p ima y objec i es o ou s udy we e (1) o in es iga e he ac o ially-combined
e ec s o inc eased sp ing ain all a iabili y and inc eased mowing equency on he
p oduc i i y and he o age quali y o semi-na u al, Cen al-Eu opean empe a e g assland and
(2) o de e mine, whe he mowing equency ampli ies o bu e s he e ec s o ain all
a iabili y on biomass p oduc ion and lea quali y o a a ge species. We conduc ed a ield
expe imen in which we al e ed he empo al dis ibu ion and he magni ude o he ain all
e en s, bu no he o e all ain all sum. To assess po en ial in e ac ions be ween ain all
Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency
148
a iabili y and mowing equency, we c ossed he ac o ain all a iabili y wi h he ac o
mowing equency ( wo o ou imes pe yea ). In he p e ious yea , we al e ed he o al
ain all amoun s along wi h he al e a ions in ain all a iabili y. This enables a compa ison
be ween he e ec s o he al e ed o al ain all amoun s and dis ibu ion and he e ec s o
al e ed ain all a iabili y unde cons an o al ain all amoun s.
We hypo hesized ha
(i) inc eased ain all a iabili y nega i ely a ec s p oduc i i y and lea quali y, as has been
shown o o he mesic g asslands,
(ii) inc eased ain all a iabili y alone can cause changes in p oduc i i y ha a e compa able
o changes caused by al e a ions in bo h, a iabili y and he annual sum o ain all oge he ,
(iii) mo e equen mowing inc eases p oduc i i y and o age quali y, as has been shown o
mo e equen , bu s ill mode a e mowing equencies,
(i ) mo e equen mowing bu e s ad e se e ec s o inc eased ain all a iabili y on
p oduc i i y and lea quali y, as g ow h esponses migh be synch onized and less esponsi e
o ain all changes a e mowing.
2. Ma e ial & Me hods
2.1 S udy si e
The s udy was conduc ed wi hin he EVENT II expe imen in a semi-na u al
g assland in he Ecological Bo anical Ga den o he Uni e si y o Bay eu h, Ge many, Cen al
Eu ope (49°55´19´´N, 11°3455``E, 365 m asl) (Jen sch & Beie kuhnlein, 2010). Communi ies
a e domina ed by all g asses, especially Alopecu us p a ensis L. (meadow ox ail). The
egional clima e is empe a e and mode a ely con inen al, wi h a mean annual empe a u e o
8.2 °C (1971–2000), and daily means anging be ween -19.6 and 27.6. The mean annual
p ecipi a ion o 724 mm (1971–2000) has a bimodal dis ibu ion wi h a majo peak in
June/July and a second peak in Decembe /Janua y (da a: Ge manWea he Se ice). The
expe imen was ins alled on a semi-na u al, es ablished meadow. Fo mo e han 20 yea s p io
o he expe imen , he meadow was mown wice pe yea and no e ilized. The ec angula ly
shaped expe imen al a ea has a o al heigh di e ence o 95 cm wi hin he diagonal om
sou hwes o no h eas , and abou 7 cm om sou heas o no h wes .
The soil o he expe imen is classi ied as S agnosol wi h a sandy-loamy Ap-ho izon
o abou 30 cm dep h, a s ongly loamy Sw-ho izon (20 cm) and a sandy-clayey Sd-ho izon
(>40 cm). Plan oo s mainly occu in he uppe 15 cm, wi h almos no oo s pene a ing
below he A-ho izon, mean pH- alue is 5.9.
Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency
149
2.2 Expe imen al Design
The EVENT II expe imen was es ablished in 2008. The expe imen al design consis s
o wo ac o ially-c ossed ac o s: (1) manipula ion o he empo al dis ibu ion and
magni ude o ain all e en s in he g owing season and (2) manipula ion o mowing
equency. We implemen ed h ee scena ios o ain all a iabili y ea men s in 2008 and
2009, assigned o he same plo s: (1) low ain all a iabili y wi h weekly i iga ion, ensu ing a
con inuous wa e supply, (2) in e media e ain all a iabili y, wi h na u al ambien ain all
a iabili y and (3) ex eme ain all a iabili y, including an ex eme sp ing d ough .
Table 1 A e age soil mois u e [ ol %], a ia ion coe icien (CV) o soil mois u e [%], numbe o ain all
e en s exceeding 1 mm, he sum o he ain all amoun [mm] and he a ia ion coe icien (CV) o daily
ain all amoun [%] in 2008 and 2009.
pa ame e yea ime span low mid ex eme na u al
a e age soil mois u e 2008 26/05-30/10 2008 (158 days) 29 21 19
2009 01/04-31/10 2009 (214 days) 30 29 25
2009 01/04-17/05 2009 (47 days) 42 40 36
2009 -29/06 2009 (43 days) 29 29 21
2009 -09/08 2009 (43 days) 32 31 27
2009 -28/10 2009 (80 days) 23 24 22
CV soil mois u e 2008 26/05-30/10 2008 (158 days) 20 31 38
2009 01/04-31/10 2009 (214 days) 35 35 37
2009 01/04-17/05 2009 (47 days) 6 8 13
2009 -29/06 2009 (43 days) 21 24 30
2009 -09/08 2009 (43 days) 25 29 26
2009 -28/10 2009 (80 days) 35 36 35
no. o e en s 2008 26/05-30/10 2008 (158 days) 61 53 45
2009 01/04-31/10 2009 (214 days) 80 71 60
2009 01/04-17/05 2009 (47 days) 19 16 16
2009 -29/06 2009 (43 days) 17 13 1
2009 -09/08 2009 (43 days) 17 16 17
2009 -28/10 2009 (80 days) 27 25 25
p ecipi a ion sum 2008 26/05-30/10 2008 (158 days) 445.2 334.8 296.1 334.8
2009 01/04-31/10 2009 (214 days) 596.8 596.8 596.8 458.5
2009 01/04-17/05 2009 (47 days) 130.2 99.5 99.5 99.5
2009 -29/06 2009 (43 days) 102.5 108.1 36.6 77.4
2009 -09/08 2009 (43 days) 164.2 152.2 223.7 127.1
2009 -28/10 2009 (80 days) 199.9 205 205 154.5
CV p ecipi a ion 2008 26/05-30/10 2008 (158 days) 164 183 204 183
2009 01/04-31/10 2009 (214 days) 204 256 297 227
2009 01/04-17/05 2009 (47 days) 227 280 280 280
2009 -29/06 2009 (43 days) 156 248 656 183
2009 -09/08 2009 (43 days) 192 205 223 220
-28/10 2009 (80 days) 203 275 275 192
1 Highes alues in each ca ego y a e in bold.
2 Values o he ege a ion pe iod 2008 and 2009 and o he ime spans be ween he compensa ion i iga ion ea men s in 2009 o he
di e ing ain all a iabili y ea men s a e gi en. Values o 2009 shown o e one ime span begin wi h a compensa ion i iga ion and
exclude he ollowing compensa ion i iga ion, as he la e is only e ec i e o soil mois u e and biomass o he ollowing pe iod.
Manusc ip 6: Inc eased ain all a iabili y educes biomass and o age quali y o empe a e g assland
la gely independen o mowing equency
150
In 2008, he i s yea o he s udy, o al g owing season amoun o ain all and
a iabili y o ain all we e al e ed. This made i possible o assess di ec d ough e ec s, as
he ex eme ain all a iabili y ea men also ecei ed leas o al ain all (see Table 1 o an
o e iew o e soil mois u e and ain all pa ame e s in bo h yea s).
In 2009, he main yea o he s udy, we con olled he amoun o ain all o e he
g owing season o all ea men s and manipula ed only he dis ibu ion o ain all, in o de o
isola e he e ec o ain all a iabili y. All ain all a iabili y ea men s we e adjus ed o he
o al 597 mm o ain all o he low a iabili y ea men in ou compensa ion i iga ions
(Table 2). Thus, no only he leng h o he d y in e als, bu also he magni ude o ain all pe
e en was changed.
The low ain all a iabili y ea men ecei ed a leas he 30-yea weekly a e age
ain all each week. The ege a ion pe iods om 1971 o 2000 se ed as a e e ence (da a:
Ge man Wea he Se ice). Missing amoun s on na u al ain all we e added i he weekly
ain all was less han he long- e m a e age o he same week. This ea men ensu ed
con inuous wa e a ailabili y. I weekly ain all exceeded he long- e m sum, i was no
sub ac ed o he nex i iga ion. Fo 2008, he o e all ain all amoun o 553 mm on he low
ain all a iabili y ea men (na u al plus i iga ed ain all) wi hin he ege a ion pe iod
(Ap il 1s -Oc obe 30 h) exceeded he 30-yea -a e age by 94 mm. In 2009, he o al amoun o
597 mm, i iga ed on all ea men s by applying compensa ion i iga ions (see below),
exceeded he 30-yea -a e age by 138 mm. Bo h yea s consequen ly esemble a he we
yea s.
The in e media e ain all a iabili y ea men emained unde ambien condi ions
wi hou any ea men , excep o he compensa ion i iga ions applied in 2009 which adjus ed
ain all sum o he low ain all a iabili y ea men a ou poin s o ime (Table 2). Thus, in
2009 he in e media e ain all a iabili y ea men ecei ed he ambien ain all plus he
compensa ion i iga ions.
Table 2 Amoun and iming o compensa ion i iga ion [mm] in 2009 on he ex eme and in e media e
ain all a iabili y ea men s gi en o apply he same o e all ain all amoun on all ea men s o e he
ege a ion pe iod.
T ea men
da e ex eme mid
May 18 h 36.6 36.6
June 30 h 96.5* 25.1
Augus 10 h 60.3 60.3
Oc obe 28 h 32 32
*applied on wo consecu i e days