pls-07-00349 Ma ch 25, 2016 Time: 11:51 # 1
ORIGINAL RESEARCH
published: 30 Ma ch 2016
doi: 10.3389/ pls.2016.00349
Edi ed by:
Bo is Rewald,
Uni e si y o Na u al Resou ces
and Li e Sciences, Vienna, Aus ia
Re iewed by:
Yao-Bin Song,
Hangzhou No mal Uni e si y, China
Bi-Cheng Dong,
Beijing Fo es y Uni e si y, China
*Co espondence:
Se gio R. Roiloa
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Func ional Plan Ecology,
a sec ion o he jou nal
F on ie s in Plan Science
Recei ed: 08 Janua y 2016
Accep ed: 07 Ma ch 2016
Published: 30 Ma ch 2016
Ci a ion:
Roiloa SR, Re ue o R, Campoy JG,
No oa A and Ba ei o R (2016)
Di ision o Labo B ings G ea e
Bene i s o Clones o Ca pob o us
edulis in he Non-na i e Range:
E idence o Rapid Adap i e
E olu ion. F on . Plan Sci. 7:349.
doi: 10.3389/ pls.2016.00349
Di ision o Labo B ings G ea e
Bene i s o Clones o Ca pob o us
edulis in he Non-na i e Range:
E idence o Rapid Adap i e
E olu ion
Se gio R. Roiloa1*, Rubén Re ue o2, Jose ina G. Campoy2, Ana No oa3,4 and
Rodol o Ba ei o1
1BioCos G oup, Depa men o Animal Biology, Plan Biology and Ecology, Facul y o Science, Uni e si y o A Co uña,
Spain, 2Uni o Ecology, Facul y o Biology, Uni e si y o San iago de Compos ela, San iago de Compos ela, Spain, 3Cen e
o In asion Biology, Depa men o Bo any and Zoology, S ellenbosch Uni e si y, Ma ieland, Sou h A ica, 4In asi e Species
P og amme, Sou h A ican Na ional Biodi e si y Ins i u e, Ki s enbosch Resea ch Cen e, Cla emon , Sou h A ica
Why some species become in asi e while o he s do no is a cen al esea ch eques in
biological in asions. Clonali y has been sugges ed as an a ibu e ha could con ibu e
o plan in asi eness. Di ision o labo is an impo an ad an age o clonal g ow h,
and i seems easonable o an icipa e ha clonal plan s may in ensi y his clonal
a ibu e in an in aded ange because o posi i e selec ion on bene icial ai s. To es
his hypo hesis, we collec ed clones o Ca pob o us edulis om na i e and in asi e
popula ions, g ew pai s o connec ed and se e ed ame s in a common ga den and
unde nega i e spa ial co a iance o nu ien s and ligh o induce di ision o labo ,
and measu ed biomass alloca ion a ios, inal biomass, and pho ochemical e iciency.
Ou esul s showed ha bo h clones om he na i e and in aded ange de elop a
di ision o labo a mo phological and physiological le el. Howe e , he bene i om
he di ision o labo was signi ican ly highe in apical ame s om he in aded ange
han in ame s om he na i e a ea. This is a no el and ou s anding esul because i
p o ides he i s e idence ha he bene i o a key clonal ai such as di ision o labo
may ha e been subjec ed o e olu iona y adap a ion in he in aded ange. The di ision
o labo can he e o e be conside ed an impo an ai in he in asi eness o C. edulis.
An app op ia e assessmen o he in luence o clonal ai s in plan in asions seems
key o unde s anding he unde lying mechanisms behind biological in asions o new
en i onmen s.
Keywo ds: biological in asions, biomass alloca ion, Ca pob o us edulis, chlo ophyll luo escence, clonal
in eg a ion, di ision o labo , local adap a ion, spec al e lec ance
INTRODUCTION
The es ablishmen o in asi e alien species modi ies he s abili y and unc ioning o popula ions,
communi ies, and ecosys ems, displaces na i e species and, as consequence, p omo es a loss o
biodi e si y (Vi ousek e al., 1996;Mack e al., 2000;S aye , 2012). In a globalized wo ld, biological
in asions and hei nega i e impac s inc eased d ama ically du ing he las decades and ep esen
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
one o he mos impo an h ea s o he conse a ion o
biodi e si y wo ldwide (Vi ousek e al., 1996;Mack e al., 2000).
The s udy o biological in asions is a apidly de eloping ield in
mode n ecology, and a c ucial eques in his esea ch a ea is o
de e mine he ai s unde lying he in asion p ocess. Howe e ,
his issue emains unsol ed (Roy, 1990;G o es and di Cas i,
1991;Lodge, 1993;Rejmánek and Richa dson, 1996;Alpe e al.,
2000;Le ine e al., 2003) and mo e e o needs o be de o ed
o iden i y he mechanisms ha explain he success o in asi e
species (Alpe e al., 2000;Le ine e al., 2003;Blackbu n e al.,
2011).
I seems easonable o assume ha some plan cha ac e is ics
migh be e explain he success o in asi e species han o he s. In
pa icula , clonal p opaga ion has been sugges ed as an a ibu e
ha could con ibu e o plan in asi eness (Pyšek, 1997;Liu e al.,
2006;Wang e al., 2008;Song e al., 2013). In ac , many o he
mos agg essi e in asi e plan species show clonal g ow h, and
a ecen s udy has highligh ed he impo ance o ai s ela ed
o clonal p opaga ion in success ul in ade s (Song e al., 2013).
Key aspec s such as physiological in eg a ion o he capaci y
o a di ision o labo make a signi ican con ibu ion o he
success o clonal plan s in a wide ange o habi a s (Ha ne
and Bazzaz, 1983;Slade and Hu chings, 1987;Alpe and S ue e ,
1997;Klimˇ
es e al., 1997;Sai oh e al., 2002;Roiloa e al., 2007).
He e, we aim o add new e idence ha may help o elucida e he
ole o clonal ai s in plan in asions.
A pi o al ask in plan in asions is o cla i y how exo ic plan s
adap o he new en i onmen s ha hey a e in ading. Th ee
mechanisms could explain he adap a ion o he in aded ange:
(i) ‘p e-adap a ion’, when species show simila ai s anda ds
in na i e and in aded anges, indica ing ha he ai is a
success ul s a egy in ei he a ea (F idley and Sax, 2014); (ii)
‘pheno ypic plas ici y’, when esponses allow plan s o adjus
hei mo phological and physiological esponses e y p ecisely o
he challenges p esen ed by pa icula en i onmen al condi ions,
p omo ing esou ce acquisi ion and success in a new habi a
(G ime and Mackey, 2002;Vallada es e al., 2007;Momme
e al., 2011); and (iii) ‘local adap a ion’, whene e he e is a apid
adap i e e olu ion due o new selec ion p essu es, esul ing in
imp o ed i ness in he in oduced en i onmen (Ma on e al.,
2004;Cano e al., 2008;Xu e al., 2010;Buswell e al., 2011).
Common ga den expe imen s compa ing plan s om na i e
and in aded anges a e equi ed o es whe he ai shi s a e
due o pheno ypic plas ici y o local adap a ion (Wol e e al.,
2004;E meie and B uelheide, 2005;Güsewell e al., 2006;
Zou e al., 2007). T ai di e ences be ween popula ions om
na i e and in aded anges g own alongside in a common ga den
would be indica i e o an episode o local adap a ion o he
in oduced ange. This esul would suppo he e olu ion o
in asi eness hypo hesis whe e apid gene ic changes a e d i en
by na u al selec ion p essu es in he in aded en i onmen (Lee,
2002;S ockwell e al., 2003).
One o he mos s iking a ibu es o clonal g ow h is he
capaci y o a ‘di ision o labo ’ (i.e., specializa ion o acqui e
locally abundan esou ces, he eby inc easing clone’s o e all
pe o mance). S olon and hizome in e nodes allow esou ce
sha ing be ween he connec ed ame s o a clonal sys em, which
he e o e a e physiologically in eg a ed. Resou ces a e gene ally
ans e ed om ame s g owing unde condi ions o high
esou ce supply o ame s loca ed in a eas whe e esou ce supply
is low, ollowing a sou ce-sink sys em (Ha ne and Bazzaz,
1983;Roiloa and Re ue o, 2005). As esul o his in eg a ion,
suppo ed ame s bene i in e ms o g ow h and su i al
(e.g., Ha ne and Bazzaz, 1983;Slade and Hu chings, 1987;
Sai oh e al., 2002;Roiloa and Re ue o, 2006). He e ogeneous
dis ibu ion o essen ial esou ces, a spa ial and empo al
scale, is a cha ac e is ic o many na u al en i onmen s (Chapin
e al., 1987;Lechowicz and Bell, 1991;Caldwell and Pea cy,
1994). In his sense, na u al habi a s posses bo h a o able and
un a o able pa ches which a e o en nega i ely co ela ed in he
space (S ue e and Hu chings, 1994). This pa chy dis ibu ion
o esou ces commonly conduc s o si ua ions in which he
esou ce-acqui ing s uc u es o clonal plan occupy si es ha
di e in quali y (Hu chings and Wijesinghe, 1997). When he
a ailabili ies o wo essen ial esou ces a e nega i ely co ela ed in
space, physiological in eg a ion can induce a plas ic esponse in
which one ame specializes o acqui e he esou ce ha is locally
abundan o i bu sca ce o o he ame s (F iedman and Alpe ,
1991;Bi ch and Hu chings, 1994;S ue e e al., 1996;Alpe
and S ue e , 1997). Because he esou ce acquisi ion is expec ed
o be mo e economical a high concen a ions, he subsequen
ecip ocal ans e o esou ces be ween ame s should inc ease
he o e all pe o mance o he clone (S ue e e al., 1996;Alpe
and S ue e , 1997;Hu chings and Wijesinghe, 1997;S ue e ,
1998). This specializa ion o acqui e locally abundan esou ces
is e med ‘di ision o labo ’ (Alpe and S ue e , 1997;Hu chings
and Wijesinghe, 1997;S ue e , 1998), and appea s o be a singula
ai o clonal species. In compa ison, non-clonal plan s o
disconnec ed ame s in a clonal sys em, ypically espond o
esou ces a ailabili y by ollowing he op imal pa i ioning heo y
ha p edic s an inc ease in he ela i e alloca ion o biomass
o s uc u es specialized in acqui ing he mos limi ing esou ce
(Tho nley, 1972;Bloom e al., 1985).
Plan in ade s usually show as e g ow h a es in he
in oduced ange han in he na i e a ea (El on, 1958;Lege and
Rice, 2003;Jakobs e al., 2004;Bossdo e al., 2005). Di ision
o labo could be accep ed as an impo an ad an age o clonal
p opaga ion, bo h a he na i e and in aded ange. Howe e ,
as he in asion p ocess can be conside ed as a con inuum wi h
a numbe o il e s ha he success ul in ade mus o e come
(Richa dson e al., 2000), i seems easonable o an icipa e ha
clonal plan s may in ensi y his clonal a ibu e in he in aded
ange gi en a posi i e selec ion on bene icial ai s, esul ing in
a apid adap i e e olu ion o his ai (Roiloa e al., 2015).
To es his hypo hesis, we (i) collec ed clones o Ca pob o us
edulis om na i e and in asi e popula ions, (ii) g ew pai s o
connec ed and se e ed ame s in a common ga den expe imen ,
unde nega i e spa ial co a iance o nu ien s and ligh , in
o de o induce di ision o labo , and (iii) measu ed biomass
alloca ion a ios, inal biomass, and pho ochemical e iciency o
quan i y ame specializa ion and pe o mance a mo phological
and physiological le el. Ou speci ic hypo hesis is ha he
capaci y o di ision o labo , and consequen bene i , would be
g ea e in popula ions om he in aded ange (i.e., Po ugal and
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
Spain) han in hose om he na i e ange (i.e., Sou h A ica).
Thus, we p edic ha (i) connec ion would induce an inc ease
in p opo ional oo mass and a dec ease in pho ochemical
e iciency in ame s unde low ligh and high nu ien s, and
ha his specializa ion o acqui e below-g ound esou ces will
be g ea e in ame s om he in aded ange han in hose
om he na i e ange; (ii) connec ion would induce a dec ease
in p opo ional oo mass and an inc ease in pho ochemical
e iciency in ame unde high ligh and low nu ien s, and ha
his specializa ion o acqui e abo e-g ound esou ces will be
g ea e in ame s om he in aded ange han in ame s om
he na i e ange; and (iii) he subsequen ecip ocal ans e
o esou ces be ween connec ed ame s should inc ease o e all
pe o mance o he clone, and his bene i would be g ea e in
clones om he in aded ange han in clones om he na i e
ange.
Many expe imen s ha e compa ed ela i e pe o mance in
in asi e and na i e o exo ic non-in asi e species (Pyšek and
Richa dson, 2007;Ga cia-Se ano e al., 2009). None heless,
ela i ely ew s udies ha e de e mined whe he in ade s change
hei unc ional s a egies om he na i e o he in oduced ange
(Zou e al., 2007;Leishman e al., 2014;Hebe ling e al., 2015).
Ou s udy appea s o be he i s in es ing hese di e ences o a
key clonal ai such as di ision o labo in an agg essi e in ade .
Assessing he in luence o clonal ai s in plan in asions is key
o unde s anding he unde lying mechanisms behind biological
in asions and, he e o e, o p edic ing u u e in asion scena ios
as well as o designing e icien con ol s a egies in in aded
a eas. Addi ionally, his in o ma ion is likewise impo an o a
be e unde s anding o how plan s espond and e ol e in new
en i onmen s.
MATERIALS AND METHODS
S udy Species
Ca pob o us edulis (L.) N.E. B ., is a ma - o ming succulen
clonal plan , na i e o he Cape Region (Sou h A ica), and an
agg essi e in ade in coas al ecosys ems o all he Medi e anean
clima e egions a ound he wo ld, including Aus alia, Eu ope
and Ame ica (D’An onio and Mahall, 1991;T a ese e al.,
2008;Vilà e al., 2008). C. edulis o ms dense ma s and sp eads
ho izon ally by he p oduc ion o nume ous apical ame s
ha emain physiologically in eg a ed by s olon connec ions
(Wisu a and Glen, 1993). Rame s p oduce oo s a e di ec
con ac wi h he subs a e and can su i e i disconnec ion
om he pa en ame occu s. This ype o ege a i e g ow h
allows C. edulis a e y e ec i e coloniza ion o he su ounding
a ea, compe ing agg essi ely wi h local species and a ec ing
nega i ely he di e si y o he na i e lo a (D’An onio and Mahall,
1991;T a ese e al., 2008). P e ious expe imen s ha e s udied
se e al aspec s o he ecology o C. edulis as plan –pollina o
ne wo ks, plan –soil eedbacks, o hyb idiza ion s udies (Vilà and
D’An onio, 1998;Ba omeus e al., 2008;de la Peña e al., 2010).
Howe e , he impo ance o clonal ai s in he expansion o his
agg essi e in ade has been gene ally o e looked (bu see Roiloa
e al., 2010, 2013, 2014a,b).
Sampling P o ocol
Fou spa ially sepa a ed popula ions o C. edulis we e sampled in
he na i e (Cape Region, Sou h A ica) and ou in he in aded
ange (Ibe ian Peninsula, Sou h Eu ope) (see Figu e 1). Plan
ma e ial was collec ed in he na i e and in he in aded ange om
coas al sand dune sys ems whe e C. edulis ypically inhabi s. To
ha e a mo e comp ehensi e illus a ion o he gene ic a iabili y,
we selec ed 36 sepa a ed clumps in each o he eigh popula ions
sampled. Wi hin a popula ion, selec ed clumps we e sepa a ed
a leas 25 m om he o he s. C. edulis o ms compac clumps
(Wisu a and Glen, 1993) and i is easonable o assume ha
each sepa a ed clump ep esen s a di e en geno ype. Howe e ,
no gene ic analyses we e pe o med and he clumps migh o
migh no di e in geno ype. Fou -membe un- oo ed clonal
agmen s we e excised a he edge o each clump. Clonal
agmen s con ained he i s ou uni s o modules ( ame s sensu
Ha pe , 1977) om he apices, and hus we ensu ed ha all
he expe imen al plan ma e ial had he same de elopmen al
s age. Plan ma e ial om he na i e ange was collec ed a
mid-Janua y 2015 and main ained in he g eenhouse du ing
3 mon hs be o e he expe imen began o minimize ma e nal
en i onmen al e ec s.
Expe imen al Design
In Ap il 2015, 48 ame pai s comp ised by he hi d and ou h
ame s om he apices we e selec ed o size uni o mi y om
he plan s ock. Ini ial size o ame pai s was es ima ed by
esh mass. P elimina y analysis showed ha he ini ial sizes
o ame pai s om he na i e and he in aded ange did
no di e signi ican ly (ANOVA F1,46 =1.787, P=0.188).
The expe imen al design consis ed o wo c ossed ac o s wi h
egion (na i e, in aded) and connec ion (connec ed, se e ed)
as main ac o s. The egion ac o included ame pai s om
coas al sand dunes o he na i e (Sou h A ica) and in aded
ange (Eu ope), as explained abo e. In he connec ion ac o ,
ame s wi hin each pai we e ei he le connec ed (di ision o
labo allowed) o se e ed (di ision o labo p e en ed). Rame s
we e se e ed by cu ing he connec ing s olon hal way be ween
hem. We did no obse e any immedia e nega i e side e ec
o cu ing he s olon (e.g., sudden dea h o disease). F om he
36 clumps sampled in each popula ion, we andomly selec ed 6
clumps o ob ain he 48 ame pai s used in he expe imen (8
popula ions ×6 clumps). Rame pai s om each o he eigh
popula ions sampled in he ield we e equally ep esen ed and
andomly assigned o each combina ion o egion by connec ion
ea men s.
Each pai was subjec ed o a egime o esou ce a ailabili y
known o induce di ision o labo ia changes in alloca ion o
mass be ween oo s and shoo s (Roiloa e al., 2014b). Olde
ame s ( he “ ou h” modules) we e subjec ed o low ligh
and high nu ien condi ions, and younge ame s ( he “ hi d”
modules) we e exposed o high ligh and low nu ien s condi ions
(see Figu e 2). This egime o esou ce a ailabili y mimics he
na u al condi ions o C. edulis g owing a coas al sand dune
habi a s, whe e olde ame s usually g ow shaded by sh ubs,
which en ich he nu ien con en o he soil, whe eas de eloping
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
FIGURE 1 | Si e loca ion (la i ude and longi ude) o Ca pob o us edulis popula ions sampled om he na i e (Cape Region in Sou h A ica) and he
in aded (Po ugal and Spain in Eu ope) egions. (SA, Sou h A ica; PT, Po ugal; SP, Spain).
younge ame s sp ead in o he non-shaded sand wi h low
nu ien con en (S. R. Roiloa, pe sonal obse a ion). High
nu ien condi ions consis ed o a 3:1 mix u e o po ing compos
and sand. In he low nu ien condi ions, ame s g ew in sand.
Low ligh condi ions we e c ea ed using a polyp opylene shade
clo h ha educes ambien ligh o 10%. Rame s in he high ligh
ea men we e le unshaded. Each pai o ame was plan ed
in a single 5L plas ic po he me ically di ided in o wo equal
compa men s by plas ic ba ie s o a oid oo in e ac ions (see
Figu e 2). None o he ame s had oo s a he s a o he
expe imen . Each ea men was eplica ed 12 imes (n=12).
The common ga den expe imen was ca ied ou in a open-
end g eenhouse a he Uni e si y o San iago de Compos ela
(Spain) (42◦52026.6500 N, 8◦33031.6400 W). In o de o a oid
con ounding e ec o posi ion wi hin he g eenhouse, he ou
ypes o ea men s we e in e spe sed andomly. Plan s we e
wa e ed egula ly wi h as much wa e as necessa y o main ain
soil mois u e. T ea men s began on 10 Ap il 2015 and con inued
o 90 days.
Measu emen s
Spec al Re lec ance
Lea spec al e lec ance pa ame e s we e measu ed 30, 60, and
90 days a e ea men applica ion using a po able spec ome e
(UniSpec Spec al Analysis Sys em, PP Sys ems, Ha e hill, MA,
USA). Speci ically, we de e mined he pho ochemical e lec ance
index (PRI), ha was calcula ed as (R539−R570)/(R539 +R570),
whe e R539 and R570 a e e lec ances a 539 and 570 nm,
espec i ely (Filella e al., 1996). This index co ela es wi h bo h
ne CO2up ake and pho osyn he ic adia ion-use e iciency (mol
CO2/mol pho ons) (Peñuelas e al., 1995;Filella e al., 1996;
Gamon e al., 1997).
Chlo ophyll Fluo escence
Immedia ely a e e lec ance measu emen s, chlo ophyll
luo escence pa ame e s we e de e mined by he sa u a ion
pulse me hod (Sch eibe e al., 1998) using a po able pulse-
ampli ude-modula e luo ome e (MINI-PAM pho osyn hesis
yield analyse ; Walz, E el ich, Ge many). In pa icula , we
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FIGURE 2 | Scheme o he expe imen , showing le els o ligh and
nu ien s gi en o pai s o connec ed o se e ed ame s. Olde ame s
we e exposed o low ligh and high nu ien condi ions. Younge ame s we e
exposed o high ligh and low nu ien condi ions. See ex o expe imen al
design de ails.
measu ed he maximum quan um yield o pho osys em II (PSII),
F /Fm=(Fm– F0)/Fm, whe e Fmand F0a e he maximum
and minimum luo escence yield, espec i ely, o da k-adap ed
samples, a e a sa u a ion pulse (>5000 µmol pho ons m−2s−1
o ac inic whi e ligh ) (see Bolhà -No denkamp e al., 1989).
The maximum PSII quan um yield (F /Fm) cha ac e izes
he pho osyn he ic p ocess associa ed wi h elec on anspo
(ligh eac ions), and p o ides in o ma ion on he e iciency o
exci a ion ene gy cap u e by open PSII eac ion cen es (Bu le
and Ki ajima, 1975). F /Fmco ela es wi h he amoun o ca bon
gained pe uni o ligh abso bed (Bolhà -No denkamp and
Öquis , 1993). This a iable was measu ed a e a 30-min da k
adap a ion pe iod, which allowed he PSII eac ion cen e s o he
lea o be ully open.
G ow h
A he end o he expe imen , each ame was sepa a ed in o
shoo s (including lea es and s olons) and oo s, d ied a 80◦C o
72 h, and weighed. The o al d y mass (shoo d y mass + oo
d y mass) and he p opo ional biomass alloca ed o oo s ( oo -
shoo a io, RSR = oo d y mass/shoo d y mass) we e calcula ed
o olde and younge ame s sepa a ely. To al d y mass a whole
clone le el (olde +younge ame s) was also calcula ed.
S a is ical Analysis
P io o analyses, a iables we e ans o med as necessa y o
mee he assump ions o pa ame ic es s. Thus, he oo /shoo
(RSR) o olde ame s we e squa e oo ans o med. We
analyzed di e ences in he o al d y mass and he p opo ional
biomass alloca ed o oo s (RSR) by wo-way analysis o
a iance (ANOVA) wi h egion (na i e, in aded) and connec ion
(connec ed o se e ed) as ixed e ec s. Sepa a es analyses
we e conduc ed o olde (wi h low ligh and high nu ien s)
and younge (wi h high ligh and low nu ien s) ame s.
Simila ly, o al d y mass a whole clone le el (olde +younge
ame s) was compa ed by wo-way ANOVA wi h egion and
connec ion as main ac o s. Changes in lea spec al e lec ance
(PRI) and chlo ophyll luo escence (F /Fm) o e ime we e
analyzed wi h wo-way analyses o a iance wi h epea ed
measu es (ANOVAR), using egion and connec ion as be ween-
subjec e ec s. Fo hese a iables, we conduced sepa a es
analyses o olde and younge ame s. Th ee ame s (one
olde and wo younge ) died du ing he expe imen , and he
co esponding pai s (olde +younge ) we e excluded om he
TABLE 1 | Resul s o wo-way analyses o a iance (ANOVA) o examine he e ec s o egion and connec ion on oo o shoo a io (RSR) o he olde and
younge ame s.
Roo /shoo (RSR) To al d y mass
E ec d F P d F P
Olde ame
Region 1 0.127 0.723 1 0.318 0.576
Connec ion 1 163.595 <0.001 1 2.089 0.156
Region ×connec ion 1 0.229 0.635 1 12.498 0.001
E o 41 41
Younge ame
Region 1 0.898 0.349 1 3.817 0.058
Connec ion 1 28.124 <0.001 1 107.025 <0.001
Region ×connec ion 1 0.122 0.729 1 4.894 0.033
E o 41 41
Whole clone
Region nd nd nd 1 1.135 0.293
Connec ion nd nd nd 1 63.965 <0.001
Region ×connec ion nd nd nd 1 0.111 0.740
E o nd 41
ANOVAs o de ec e ec s o ea men s on o al d y mass o olde and younge ame s, and a whole clone le el (olde +younge ame s). nd, non-de e mined. Values o
P<0.05 a e in bold ace. See Figu es 3 and 4 o da a.
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
FIGURE 3 | Final measu emen s (mean +SE) o he p opo ional d y mass alloca ed o oo s (de e mined as he oo o shoo a io, RSR) and he o al
d y mass (g) o connec ed and se e ed olde (le ) and younge ( igh ) ame s om na i e and in aded egions. See Table 1 o ANOVA esul s.
analyses. This educed he numbe o eplica es used in he
di e en analyses, as indica ed by he e o deg ee o eedom.
Signi icance le el was se a P<0.05. S a is ical es s we e
pe o med wi h SPSS S a is ics 19.0 (IBM, A monk, New Yo k,
USA).
RESULTS
G ow h
The p opo ion o biomass alloca ed o oo s by olde and
younge ame s, as de e mined by he oo o shoo a io (RSR),
was signi ican ly a ec ed by he connec ion ea men (Table 1).
Connec ion signi ican ly inc eased he p opo ion o d y mass
alloca ed o oo s (RSR) in olde ame s bu i was dec eased
in younge ame s (Figu e 3). The e ec o egion and he
in e ac ion be ween egion and connec ion on oo o shoo
(RSR) was no signi ican nei he in olde no in younge ame s
(Table 1).
The inal o al d y mass o olde ame s was signi ican ly
a ec ed by he in e ac ion be ween egion and habi a (Table 1).
Connec ion signi ican ly inc eased he o al d y mass in he
geno ypes om he na i e egion. Howe e , his e ec was no
main ained in he geno ypes om he in aded egion, whe e we
FIGURE 4 | Final measu emen s (mean +SE) o he o al d y mass (g)
o he whole clones (olde +younge ame s) om he na i e and
in aded egions in he connec ed and se e ed ea men s. See Table 1
o ANOVA esul s.
de ec ed a dec ease o he o al d y mass in he connec ed olde
ame s (Figu e 3). On he o he hand, connec ion signi ican ly
inc eased he inal d y mass o younge ame s (Table 1;
Figu e 3). This posi i e e ec o connec ion on he inal d y mass
o younge ame s was s onge in geno ypes om he in aded
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
egion, as deno ed by he signi ican e ec o he in e ac ion
be ween ac o s egion and connec ion (Table 1;Figu e 3).
This esul indica es ha he bene i o physiological in eg a ion
was signi ican ly highe in in asi e popula ions han in na i e
popula ions. A he whole clone le el (olde +younge ame s),
ou esul s showed a signi ican e ec o connec ion, wi h an
inc ease o he inal o al d y mass due o connec ion bo h in
he geno ypes om he na i e and he in aded egion (Table 1;
Figu e 4). Nei he he egion no he in e ac ion be ween egion
and connec ion had a signi ican e ec on o al d y mass a he
whole clone le el (Table 1).
Lea Spec al Re lec ance and Chlo ophyll
Fluo escence
Olde ame s om he in aded egion showed a signi ican ly
highe PRI han olde ame s om he na i e egion (Table 2;
Figu e 5). Ou esul s also showed a signi ican e ec o he
connec ion ea men on he PRI o olde ame s (Table 2).
Thus, he e was a signi ican dec ease in PRI o connec ed
olde ame s, ega dless o whe he hey came om he na i e
o he in aded egion (Figu e 5). We de ec ed a signi ican
e ec o he in e ac ion be ween egion and connec ion in he
maximum quan um yield o pho osys em II (F /Fm) o olde
ame s (Table 2). Connec ion signi ican ly inc eased F /Fmin
olde ame s o he geno ypes om he na i e egion, bu
dec eased i in he olde ame s o geno ypes om he in aded
egion (Figu e 5).
Connec ion signi ican ly a ec ed he PRI o younge ame s
(Table 2). PRI alues we e signi ican ly highe in connec ed han
in se e ed younge ame s bo h in geno ypes om he na i e
and he in aded egion (Figu e 5). Al hough we did no de ec
a signi ican be ween-subjec e ec o connec ion in younge
ame s in e ms o maximum quan um yield o pho osys em II
(F /Fm) (Table 2;Figu e 5), his a iable was signi ican ly a ec ed
by he in e ac ion o connec ion by ime (wi hin-subjec e ec )
(Table 2). Thus, he F /Fm alues o younge ame s changed
wi h ime, and his change was dependen on he connec ion
ea men . Connec ed and se e ed younge ame s signi ican ly
in e ed hei F /Fm alues du ing he expe imen , ega dless
o hei egion o o igin. A day 30 F /Fm alues we e highe
in connec ed han in se e ed younge ame s, whe eas se e ed
younge ame s showed g ea e F /Fm alues han connec ed
ame s a days 60 and 90 (Figu e 6).
DISCUSSION
Ou esul s suppo he exis ence o di ision o labo , bo h a
mo phological and a physiological le el, in na i e and in aded
TABLE 2 | Resul s o wo-way epea ed-measu e analysis o a iance (ANOVAR) wi h egion and connec ion as be ween-subjec e ec s, o di e ences
in he pho ochemical e lec ance index (PRI) and he maximum quan um yield o pho osys em PSII (F /Fm) o olde and younge ame s.
PRI F /Fm
E ec d F P d F P
Olde ame
Be ween-subjec e ec s
Region 1 4.933 0.032 1 0.135 0.715
Connec ion 1 7.933 0.007 1 0.230 0.634
Region ×connec ion 1 0.615 0.437 1 5.994 0.019
E o 41 41
Wi hin-subjec e ec s
Time 2 41.226 0.001 2 17.732 0.001
Region × ime 2 2.154 0.123 2 1.081 0.344
Connec ion × ime 2 0.879 0.419 2 0.012 0.988
Region ×connec ion × ime 2 0.408 0.666 2 1.049 0.355
E o 82 82
Younge ame
Be ween-subjec e ec s
Region 1 0.008 0.928 1 1.315 0.258
Connec ion 1 4.635 0.037 1 0.579 0.451
Region ×connec ion 1 0.397 0.532 1 0.305 0.584
E o 41 41
Wi hin-subjec e ec s
Time 2 18.015 0.001 2 22.316 0.001
Region × ime 2 0.012 0.988 2 0.154 0.857
Connec ion × ime 2 0.853 0.430 2 5.749 0.005
Region ×connec ion × ime 2 1.62 0.204 2 0.220 0.803
E o 82 82
Values o P <0.05 a e in bold ace. See Figu es 5 and 6 o da a.
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
FIGURE 5 | Mean alues (+SE) o pho ochemical e lec ance index (PRI) and he maximum quan um yield o pho osys em II (F /Fm) o connec ed
and se e ed olde (le ) and younge ( igh ) ame s om he na i e and in aded egions. See Table 2 o ANOVA esul s.
popula ions o C. edulis. As p oposed in ou hypo heses, we
ound ha connec ion signi ican ly inc ease he mass alloca ed
o oo s (RSR) in olde ame s subjec ed o high nu ien s
and low ligh condi ions, deno ing a specializa ion o acqui e
he ela i ely abundan below-g ound esou ce. On he o he
hand, connec ion signi ican ly inc eased he pho osyn he ic
adia ion-use e iciency (as es ima ed by he PRI and educed
he p opo ional oo mass (RSR) in younge ame s, which we e
subjec ed o high ligh and low nu ien s condi ions, indica ing a
specializa ion o acqui e abo e-g ound esou ces. In popula ions
om bo h he na i e and he in aded ange, esou ce sha ing
media ed by s olon connec ion signi ican ly inc eased he inal
biomass a he whole clone le el. These esul s p o ide s ong
e idence ha ame s o clones o C. edulis ha e a capaci y
o di ision o labo , and ha his specializa ion o acqui ing
he mos abundan esou ce epo s a bene i o he whole
clone. Howe e , ou esul s do no suppo he p edic ion ha
clones om he in aded ange may ha e a g ea e capaci y
o di ision o labo han hose om he na i e ange. Since
esou ce acquisi ion is expec ed o be mo e economical whe e
he esou ce is mo e abundan , he specializa ion o acqui e he
ela i ely ich esou ce and he subsequen ecip ocal esou ce
sha ing be ween connec ed ame s would inc ease he o e all
pe o mance o he clone (S ue e e al., 1996;Alpe and
S ue e , 1997;Hu chings and Wijesinghe, 1997;Roiloa e al.,
2014b). Simila esul s showing a capaci y o di ision o labo
a mo phological and physiological le el we e ecen ly epo ed
by Roiloa e al. (2014b) in clones o C. edulis in he in aded
ange. Likewise, Roiloa e al. (2007) epo ed en i onmen al-
induced di ision o labo a mo phological and physiological
le els in clones o he s oloni e ous F aga ia chiloensis. Bo h
s udies showed g ea e capaci y o di ision o labo in clones
om pa chie habi a s, whe e essen ial esou ces we e nega i ely
co ela ed. In hese condi ions, he di ision o labo be ween
ame s would be specially bene icial, sugges ing an adap i e
di ision o labo induced by he en i onmen (Roiloa e al., 2007,
2014b). P e ious s udies wi h o he clonal plan s also epo ed
di e ences be ween geno ypes o se e al clonal ai s, including
di ision o labo , esou ce sha ing, o sexual/asexual shi , which
sugges a po en ial o local adap a ion (Lo sche and Hay, 1997;
Alpe , 1999;P a i and Schmid, 2000;Alpe e al., 2003;Roiloa
e al., 2007;Nilsson and D’He e eld , 2008;D’He e eld e al.,
2014), and a e in ag eemen wi h he e olu iona y heo y ha
p edic s ha popula ions e ol e o gene a e ai s o gain an
ad an age unde hei local condi ions (Willians, 1966).
Al hough ou esul s showed no di e ences in he capaci y
o di ision o labo be ween in asi e and na i e popula ions, we
ound, howe e , signi ican di e ences in he bene i s ob ained
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Roiloa e al. Adap i e E olu ion o Clonal T ai s
FIGURE 6 | Time cou se o mean alues (±SE) o pho ochemical e lec ance index (PRI) and he maximum quan um yield o pho osys em II (F /Fm)
o connec ed (solid lines) and se e ed (dashed lines) ea men s o olde (le ) and younge ( igh ) ame s om he na i e (closed symbols) and
in aded (open symbols) egions. See Table 2 o ANOVA esul s.
by he di ision o labo among ame s. As epo ed, bene i s we e
signi ican ly highe in younge ame s om in asi e popula ions
han in hose om na i e popula ions. This is a no el and
ou s anding esul because i p o ides he i s e idence ha
he bene i o a key clonal ai , as di ision o labo , may
be subjec ed o e olu iona y adap a ion in he in aded ange.
The e o e, ou esul s indica e ha apid gene ic changes linked
o selec ion p essu es migh con ibu e o he in asion o C. edulis
in he in oduced ange. Rapid adap i e e olu ion o in oduced
popula ions could explain in asion success in a new en i onmen
(Ma on e al., 2004;Sax e al., 2007). In his line, i seems
easonable o p edic ha posi i e selec ion o bene icial ai s
such as di ision o labo could be a o ing he expansion o
C. edulis in he in oduced ange and, he e o e, p omo ing i s
in asi eness.
In e es ingly, he bene i o di ision o labo de ec ed in
younge ame s a he in aded ange seems o be ob ained a
he cos o he olde ame s. This is, in na i e popula ions he
ecip ocal anspo o esou ces be ween he connec ed olde
and younge ame s seems o be balanced and, consequen ly
p o ided mu ual bene i s o bo h ame s. Howe e , ou esul s
o he in asi e popula ions showed a signi ican bene i o he
connec ion o younge ame s, bu a signi ican cos o olde
ame s, indica ing an unbalanced sha e o esou ces. This esul
seemingly indica es ha he e was a unidi ec ional anspo o
esou ces om he olde o he younge ame in he popula ion
om he in aded ange. As a esul , g ow h inc ease was
mo e p onounced in in asi e younge ame s han in na i e
ones, whe e esou ces anspo appea ed o be bidi ec ional, as
desc ibed in p e ious wo ks (e.g., S ue e e al., 1996;Alpe and
S ue e , 1997;Hu chings and Wijesinghe, 1997). This ai shi
in he in oduced ange could be p omo ing he expansion o
younge ame s, con ibu ing o he expansion o in asi e plan s.
C. edulis sp eads ho izon ally by he p oduc ion o abundan
younge ame s ha emain in eg a ed by s olon connec ions.
Roiloa e al. (2010) ound an associa ion be ween he inc ease in
o al biomass and he ho izon al expansion o apical ame s o
C. edulis when colonizing a na u al dune sys em in he in aded
ange. Thus, ou inding o an in ensi ica ion o he bene i s
de i ed om di ision o labo o apical ame s in popula ions
om in oduced ange suppo s he idea ha adap a ion a e
in oduc ion may a o he expansion o his agg essi e in ade .
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