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Spa ina e sicolo Fab e: Ano he case o Spa ina
ans-A lan ic in oduc ion?
Alex Baumel, Ma hieu Rousseau-Gueu in, C. Sapienza-Bianchi, Agnès Ga eil,
N. Duong, Hélène Rousseau, Oli ie Co i on, Rachid Ami ouche, S.
Sciand ello, B. Dua e, e al.
To ci e his e sion:
Alex Baumel, Ma hieu Rousseau-Gueu in, C. Sapienza-Bianchi, Agnès Ga eil, N. Duong, e al..
Spa ina e sicolo Fab e: Ano he case o Spa ina ans-A lan ic in oduc ion?. Biological In a-
sions, 2016, 18 (8), pp.2123-2135. �10.1007/s10530-016-1128-z�. �hal-01355664�
INVASIVE SPARTINA
Spa ina e sicolo Fab e: Ano he case o Spa ina
ans-A lan ic in oduc ion?
A. Baumel .M. Rousseau-Gueu in .C. Sapienza-Bianchi .A. Ga eil .N. Duong .
H. Rousseau .O. Co i on .R. Ami ouche .S. Sciand ello .B. Dua e .I. Cac¸ado .
J. M. Cas illo .M. Ainouche
Recei ed: 23 Ma ch 2015 / Accep ed: 20 Ma ch 2016
ÓSp inge In e na ional Publishing Swi ze land 2016
Abs ac In e con inen al in oduc ions a e wide-
sp ead in he genus Spa ina, wi h impo an ecolog-
ical and e olu iona y consequences. The na i e o
in oduced s a us o Spa ina species is hen c i ical
wi h ega d o biodi e si y assessmen , especially o
ulne able Medi e anean coas line ecosys ems. Spa -
ina e sicolo was i s eco ded in sou he n F ance in
1849, hen successi ely in a ious places on he
Eu opean and No h-A ican Medi e anean and
A lan ic coas s. This species is conside ed o be ei he
a Eu opean na i e o an in asi e species in oduced
om No h Ame ica which has a high mo phological
simila i y o he A lan ic Ame ican species Spa ina
pa ens. We pe o med ex ensi e sampling o S.
e sicolo in Eu ope and No h A ica ( om na u al
popula ions and he ba ium collec ions) and compa ed
hese samples o o he Eu opean and Ame ican
Spa ina species (including S. pa ens). Ch omosome
coun s we e epo ed o he i s ime and e ealed
ha S. e sicolo is e aploid (2n=4x=40). Phy-
logene ic analyses based on chlo oplas and nuclea
ibosomal DNA sequences did no e eal any molec-
ula a ia ion wi hin S. e sicolo . In his species, a
single haplo ype, ha is iden ical o one haplo ype o
S. pa ens, was ound in he ou chlo oplas and he
nuclea ibosomal ITS egions in es iga ed. In addi-
ion, simple sequence epea ma ke s we e used and
e ealed a low le el o gene ic di e si y wi hin S.
e sicolo , sugges ing ha he in oduc ion o S.
Gues edi o s: Alan G ay and Malika Ainouche/In asi e
Spa ina.
Elec onic supplemen a y ma e ial The online e sion o
his a icle (doi:10.1007/s10530-016-1128-z) con ains supple-
men a y ma e ial, which is a ailable o au ho ized use s.
A. Baumel N. Duong
Aix Ma seille Uni e si e
´, Ins i u Me
´di e ane
´en de
Biodi e si e
´e d’Ecologie (IMBE, UMR CNRS, IRD,
A ignon Uni e si e
´), Technopo
ˆle de l’En i onnemen
A bois-Me
´di e ane
´e, BP 80,
13545 Aix-en-P o ence Cedex 04, F ance
M. Rousseau-Gueu in C. Sapienza-Bianchi 
A. Ga eil H. Rousseau M. Ainouche (&)
UMR CNRS 6553 Ecobio, OSUR (Obse a oi e des
Sciences de l’Uni e s de Rennes), Uni e si e
´de Rennes
1/Uni e si e
´Eu ope
´enne de B e agne, 35042 Rennes,
F ance
e-mail: [email p o ec ed]
M. Rousseau-Gueu in
INRA, UMR 1349, Ins i u de Ge
´ne
´ ique, En i onnemen
e P o ec ion des Plan es, 35653 Le Rheu Cedex, F ance
O. Co i on
Pla e-Fo me de Cy oge
´ne
´ ique Mole
´culai e, INRA, UMR
1349, Ins i u de Ge
´ne
´ ique, En i onnemen e P o ec ion
des Plan es, 35653 Le Rheu Cedex, F ance
R. Ami ouche
Uni e si e
´des Sciences e de la Technologie Houa i
Boumediene, Labo a oi e de Biologie e Physiologie des
O ganismes, BP 32 El-Alia, 16111 Bab-Ezzoua , Alge ,
Alge ia
123
Biol In asions (2016) 18:2123–2135
DOI 10.1007/s10530-016-1128-z
e sicolo occu ed om a na ow gene ic pool o S.
pa ens om No h Ame ica.
Keywo ds Co dg ass Gene ic di e si y Species
s a us Medi e anean Mic osa elli es Phylogeny
In oduc ion
We land habi a s a e among he mos h ea ened in he
Medi e anean as a consequence o in ense u baniza-
ion, an h opogenic dis u bance and inc eased numbe
o in asi e axa (Me
´dail and Ve laque 1997). In
F ance, Medi e anean we lands a e among he habi-
a s ha a e he mos colonized by in asi e species
(Ve laque e al. 2002). Because su eys o biodi e si y
a e gene ally poo ly coo dina ed in he Medi e anean
biodi e si y ho spo (Ma ignani e al. 2014), in e ence
o he na i e o in oduced plan species s a us is no
i ial. This s a us is an essen ial pa ame e o
biodi e si y managemen and conse a ion biology.
I also ep esen s c i ical in o ma ion wi h ega d o
popula ion and species e olu iona y his o y. Es ab-
lishing na i e s a us o a species in a gi en egion is
no an easy ask and equi es a combina ion o
di e en app oaches o elucida e he o igin, mode o
o ma ion and biogeog aphy o he conside ed axon.
The inc eased oppo uni ies o long-dis ance human-
media ed species dispe sal make hese esea ches e en
mo e complex (Kowa ik 2003). In his con ex ,
molecula ma ke s and e olu iona y gene ics p o ide
impo an insigh o ace back popula ion, species
o igin and mig a ion his o y (Mansion e al. 2008;
Ha dion e al. 2014).
In Spa ina (co dg asses), ecu en in e con inen al
in oduc ion e en s and biological in asions a e pa ic-
ula ly common and well-documen ed (Daehle and
S ong 1996a,b;SanLe
´on e al. 1999;Baumele al.
2001;Sanche
´z-Gullon 2001; Ay es e al. 2004;Ane al.
2007;Ainouchee al.2009; Campos e al. 2004; Lona d
e al. 2010; Saa ela 2012; S ong and Ay es 2013). This
g ass genus (Poaceae, Chlo idoideae) ep esen s a well-
suppo ed monophyle ic lineage (Baumel e al. 2002;
Fo une e al. 2007) closely ela ed o some membe s o
he pa aphyle ic Spo obolus genus and Calamo il a
(Pe e son e al. 2014). I is composed o abou 15
pe ennial species ha ha e di e si ied mos ly in he
New Wo ld (Mobbe ley 1956). In oduc ion o species
ou side hei na i e ange o e he pas 150 yea s has
accele a ed di e si ica ion by acili a ing hyb idiza ion
wi h na i e species, in og ession o specia ion, esul -
ing in se e al supe imposed di e gen genomes ha
coexis in he species cu en ly ound in he wild
(Ainouche e al. 2012). The basic (haploid) ch omosome
numbe in Spa ina is conside ed o be x=10
(Ma chan 1968), and all species eco ded o da e a e
polyploid, anging om e aploids o dodecaploids.
Molecula phylogenies om nuclea and chlo oplas
DNA sequences ha e indica ed ha genus Spa ina has
e ol ed h ough wo main lineages including e aploid
and hexaploid species espec i ely (Baumel e al. 2002).
The e aploid lineage is composed o species na i e o
he New Wo ld, colonising coas al o inland sal
ma shes om ei he No he n (Spa ina pa ens,Spa -
ina bake i, Spa ina g acilis,Spa ina cynuso oides,
Spa ina pec ina a)o Sou he n(Spa ina cilia a,
Spa ina a undinacea) hemisphe es. The e aploid S.
a gen inensis (syn. S. spa inae), which has a disjunc
dis ibu ion in No h-Cen al Ame ica and in Sou h-
Ame ica, is sis e o he hexaploid lineage. This la e
clade is composed o Spa ina ma i ima,Spa ina
al e ni lo a,andSpa ina oliosa, all colonizing low
ma sh zones. Spa ina ma i ima, na i e o he Wes e n
Eu ope and A ican A lan ic coas s, isone o he ew Old
Wo ld na i e species wi h ecen axa o hyb id o igin
and he con o e sial S. e sicolo (see below). Acci-
den al o delibe a e in oduc ions lead o a ious
hyb idiza ion e en s wi hin o be ween he e aploid
and hexaploid lineages ( e iewed in Ainouche e al.
2012; S ong and Ay es 2013).
In Eu ope, in oduc ions o he hexaploid S.
al e ni lo a, na i e o he A lan ic Ame ican coas s
and i s subsequen hyb idiza ion wi h hexaploid S.
S. Sciand ello
Depa men o Biological, Geological and En i onmen al
Sciences, Uni e si y o Ca ania, ia Alongo 18,
95125 Ca ania, I aly
B. Dua e I. Cac¸ado
MARE – Ma ine and En i onmen al Sciences Cen e,
Facul y o Sciences o he Uni e si y o Lisbon, Campo
G ande, 1749-016 Lisbon, Po ugal
J. M. Cas illo
Depa amen o de Biologı
´a Vege al y Ecologı
´a, Facul ad
de Biologia, Uni e sidad de Se illa, Apa ado 1095,
41080 Se ille, Spain
2124 A. Baumel e al.
123
ma i ima led o he o ma ion o wo s e ile F1 hyb ids
in Sou he n England (S. x ownsendii) and in Sou h-
wes F ance (S. x ney au ii). Genome duplica ion in
he B i ish hyb id esul ed in he igo ous and e ile
allododecaploid S. anglica (Hubba d 1968; Gue
´ne
´gou
e al. 1988; G ay e al. 1990; G ay e al. 1991). This
species apidly expanded in ange and sp ead na u ally
o wes e n Eu opean sal ma shes. I is now in oduced
in a ious con inen s, leading o a ious a emp s o
con ol o e adica e he species (e.g. Hacke e al.
2001; Co e e al. 2007). In oduced S. al e ni lo a is
p og essing along he wes e n A lan ic coas s o
F ance and Spain (Baumel e al. 2003; Campos e al.
2004). Ano he in oduced Spa ina species in Eu ope
is he na i e Sou h-Ame ican hep aploid species S.
densi lo a (Fo une e al. 2008) ha is in ading
Medi e anean sal ma shes o he Ibe ian peninsula
(Bo olus 2006; Cas illo e al. 2008), whe e i
hyb idized wi h he hexaploid S. ma i ima (Cas illo
e al. 2010).
In he wes e n Medi e anean, damp dep essions in
dune habi a s a e colonized by Spa ina e sicolo
Fab e (Fig. 1) ha g ows also in b ackish ma shes in
he A lan ic Coas o he Sou hwes Ibe ian Peninsula.
This species, also named Spa ina juncea o Spa ina
du ieui (Che alie 1923; Sain -Y es 1932), had a
con o e sial axonomic s a us. I was ini ially
eco ded almos simul aneously in se e al Medi e -
anean places: i s in Sou he n F ance nea Agde
(Fab e 1849), hen in I aly (Pa la o e 1848–1850),
Alge ia (Cosson and Maisonneu e 1867), and Po u-
gal (Da eau 1897). In 1901, Ney au de ec ed his
axon on he Sou hwes F ench A lan ic coas , nea
A cachon (Cos e 1906). Since hen, S. e sicolo has
es ablished all along he wes e n Medi e anean coas s:
in Co sica (Jeanmonod and Bu de 1989) as well as on
he A lan ic and Medi e anean coas s o he Ibe ian
Peninsula (Sanche
´z-Gullo
´n2001). S. e sicolo was
conside ed as ei he a na i e Medi e anean plan (e.g.
Sanche
´z-Gullo
´n2001; Giuliano and S anisci 2010;
Tison e al. 2014a,b), o an in asi e species
in oduced om Ame ica (Sanz Elo za e al. 2004;
Tison and de Foucaul 2014). Based on mo phological
simila i ies, Mobbe ley (1956) conside ed S. e si-
colo as synonymous o S. pa ens, assuming ha he
Medi e anean popula ions we e in oduced om he
A lan ic No h Ame ican coas whe e S. pa ens is
abundan in high ma sh and dunes. Recen s udies on
sal ma shes along he Spanish A lan ic coas ha e
unde lined he p esence o S. pa ens in Eu ope (San
Leon 1999; Page e al. 2010) and enewed in e es in
he s a us o S. e sicolo . P ie o e al. (2011)
examined h ee S. e sicolo indi iduals om no h-
e n Spain (As u ias) using In e nal ansc ibed Space
(ITS) sequences o nuclea ibosomal DNA genes, and
compa ed hese sequences o hose ini ially published
in genus Spa ina by Baumel e al. (2002) and Fe is
e al. (unpublished). These indi iduals exhibi ed
simila ITS sequence o S. pa ens, which led hese
au ho s o sugges ha S. e sicolo should be
conside ed as S. pa ens. Bu i canno be excluded
ha a na i e Medi e anean Spa ina species exis s
besides he in oduc ion o S. pa ens in Spain. Cases o
c yp ic in asion ha e al eady been documen ed in he
ecen his o y o Spa ina (e.g. Bo olus e al. 2015)
and can be di icul o de ec (Val uena e al. 2011).
Fig. 1 Spa ina e sicolo a igo ous popula ion om Vieux Salins (Hye
` es, F ance) bmi o ic ch omosomes coun e s ained wi h
DAPI (2n =40), ba ep esen s 5 lm
Spa ina e sicolo Fab e: Ano he case o Spa ina ans-A lan ic in oduc ion? 2125
123
In his s udy, we aim a answe ing he ollowing
ques ions: Is S. e sicolo Fab e om Eu ope con-
speci ic wi h S. pa ens om No h Ame ica and i so is
Eu opean S. pa ens ano he case o ans-A lan ic
in oduc ion in o Eu ope? As highligh ed abo e,
wo ldwide in asions o Spa ina a e common phe-
nomena and he Medi e anean coas may be one o he
a ious places whe e Ame ican Spa ina ha e se led
a e dispe sal by ships du ing he 18 h o 19 h
cen u ies. Gaining insigh in o he na i e o in oduced
s a us o S. e sicolo , o i s ela ionship wi h o he
Spa ina species will be o c i ical impo ance in o de
o be e unde s and he biogeog aphy and di e si i-
ca ion o Spa ina species, as well as o de e mine he
conse a ion p io i y le el and managemen policy o
S. e sicolo in he Old Wo ld. To answe hese
ques ions, popula ions o S. e sicolo sampled om
a ious Medi e anean and A lan ic si es in Eu ope
and No h-A ica (including e e ence ypes om
he ba ia) a e analyzed using cy ogene ic and molec-
ula (mic osa elli e, nuclea and chlo oplas DNA
sequences) da a, and compa ed o No h-Ame ican
Spa ina species.
Ma e ials and me hods
Plan ma e ial
Fi y-se en indi iduals o S. e sicolo we e sampled
om nume ous Medi e anean si es (n =47) and
om he ba ium collec ions (n =10) (Table S1). The
analyzed samples include specimens om a ious
popula ions in F ance (including Co sica), I aly,
Po ugal, Spain and Alge ia as well as he i s S.
e sicolo plan s om Agde (F ance) disco e ed by
Fab e (1849). Eigh Spa ina pa ens samples we e
ob ained om he A lan ic No h Ame ican coas
(Table S1). Rep esen a i es om eigh o he Spa ina
species (S. a gen inensis,S. al e ni lo a,S. a undi-
nacea,S. bake i,S. densi lo a,S. oliosa,S. ma i ima
and S. pec ina a) and om Spo obolus c yp and us
we e addi ionally in oduced in phylogene ic analyses.
Ch omosome coun s
The ch omosome numbe o S. e sicolo was de e -
mined on mi o ic ch omosomes ob ained om wo
plan s collec ed in F ance (Vieux Salins and Sain -
Louis du Rhone). Mi o ic ch omosomes we e
obse ed on me aphasic cells isola ed om oo ips.
The oo s ips o 0.5–1.5 cm leng h we e ea ed wi h
0.04 % 8-hyd oxiquinoline o 2 h a 4 °C in he da k
ollowed by 2 h a oom empe a u e o accumula e
me aphases, hen ixed in e hanol-ace ic acid (3:1, / )
o 12 h a 4 °C and s o ed in e hanol 70 % a -20 °C.
A e washing in 0.01 M enzyme bu e (ci ic acid-
sodium ci a e pH 4.5) o 15 min, he oo s we e
diges ed in a solu ion o 5 % Onozuka R-10 cellulase
(Sigma) and 1 % Y23 pec olyase (Sigma) a 37 °C o
30 min. The oo ips we e hen washed wi h dis illed
wa e o 30 min. Roo ips ans e ed on a slide we e
squashed in a d op o 3:1 e hanol-ace ic acid ixa ion
solu ion. A e ai -d ying, slides we e s ained wi h 4,6-
diamidino-2-phenylindole (DAPI). Fluo escence
images we e cap u ed using a CoolSnap HQ came a
(Pho ome ics, Tucson, A iz) on an Axioplan 2
mic oscope (Zeiss, Obe kochen, Ge many) and anal-
ysed using Me aVue
TM
(Uni e sal Imaging Co po a-
ion, Downing on, PA).
DNA isola ion, PCR ampli ica ion and DNA
sequencing
Genomic DNA was isola ed om 100 mg o esh (o
30 mg o he ba ium) lea es om each indi idual
using he NucleoSpin
Ò
Plan II Ki (Mache ey–
Nagel), ollowing ins uc ions p o ided by he man-
u ac u e . DNA concen a ions we e es ima ed using
he Nanod op Spec opho ome e ND 1000 (The mo
Fische Scien i ic).
Fou chlo oplas and en nuclea egions we e
ampli ied. Chlo oplas sequences we e chosen among
he mos a iable in e genic egions iden i ied in
Poaceae (Rousseau-Gueu in e al. 2015) o wi hin
Spa ina (Blum e al. 2007; Kim e al. 2013): i
included he ndhC- nV, pe A-psbJ (p ime s designed
om Rousseau-Gueu in e al. 2015), and he nL- nF
and nT- nL in e genic egions (Tabe le e al. 1991).
Nuclea egions included In e nal ansc ibed Space s
(ITS) o nuclea ibosomal genes ( DNA) (Whi e e al.
1990) and nine mic osa elli e ma ke s (SSR 6, 40, 44,
72, 109, 122, 161, 172, 188) iden i ied om S.
ma i ima Bac e ial A i icial Ch omosome end
sequences (Fe ei a de Ca alho e al. 2013). The
p ime sequences used in his s udy a e indica ed in
Table S2.
2126 A. Baumel e al.
123

Ampli ica ions o chlo oplas and ITS egions we e
ca ied ou using he high ideli y KOD polyme ase
(Toyobo, No agen) in a o al olume o 50 ll. The
eac ion mix included 1X o KOD bu e , 1.5 mM
MgSO4, 0.2 mM dNTP, 0.3 lM o each p ime , 0.02
U o KOD polyme ase, and 20 ng empla e DNA.
Cycling condi ions we e 94 °C o 2 min, ollowed by
32 ounds o 94 °C o 20 s, 59.5 °C o 10 s and an
ex ension a 70 °C o 15 s. Chlo oplas and nuclea
(ITS) PCR p oduc s we e pu i ied using he PCR
Clean-up Gel ex ac ion ki (Mache ey–Nagel) and he
pu i ied p oduc s we e sen o Mac ogen Eu ope
(Ams e dam, Ne he lands) o di ec sequencing.
Long PCR p oduc s ( om he nT- nL, nL- nF
and ndhC- nV egions) we e sequenced om bo h
sides. Sequences we e cleaned and e i ied isually on
he ch oma og ams (no double peaks obse ed).
Simple Sequence Repea (SSR) de ec ion was
pe o med o 60 samples (52 Old Wo d S. e sicolo
and 8 New Wo ld S. pa ens) using 9 mic osa elli e
loci. Ampli ica ions o all mic osa elli es we e pe -
o med in 20 lL ha con ained 10 ng DNA, 4 lLo
5X bu e , 4 mM MgCl2, 0.2 mM dNTPs, 0.4 lMo
each p ime , and 0.4 uni s o Taq polyme ase (Q-
Biogen) in a PTC-200 G adien The mal Cycle (MJ
Resea ch), ollowing ouchdown PCR p o ocols
(Miglio e e al. 2013). The 50ends o he o wa d
p ime s we e labelled wi h PET o NED. The luo es-
cen ly labelled PCR p oduc s we e dilu ed (1/40) and
we e sepa a ed by capilla y elec opho esis, wi h a
500 bp size s anda d (LIZ500), using an ABI P ism
Ò
3730xl (Applied Biosys ems) au oma ic sequence .
Alleles we e sized using PEAK SCANNER 1.0
so wa e (Applied Biosys ems). Geno yping (PCR
and elec opho esis) was epea ed o 8 samples o
e i y he ep oducibili y o he peak pa e ns.
Phylogene ic analyses
The da a ma ices gene a ed o indi idual o con-
ca ena ed chlo oplas egions as well as he nuclea
ITS ma ix we e ob ained a e aligning all sequences
using Geneious (D ummond e al. 2010) and adjus ing
hem manually. These ma ices we e i s subjec ed o
phylogene ic analyses using maximum pa simony.
Sequence da a we e analyzed using PAUP* 4.0b10
(Swo o d 2001) wi h heu is ic sea ch and he de aul
sea ch op ions. The phylogene ic analyses we e pe -
o med using sequences om Spo obolus
c yp and us, a closely ela ed species o Spa ina
(Pe e son e al. 2014) as ou g oup. Boo s ap analyses
we e pe o med wi h 1000 eplica es (Felsens ein
1985). In addi ion, hese da a ma ices we e subjec ed
o Maximum Likelihood phylogene ic analyses. The
bes - i ed model o sequence e olu ion o each
egion (indi idual o conca ena ed) was de e mined
by using JModel es (Posada 2008) implemen ed in
MEGA 5.0 (Tamu a e al. 2011). Maximum likelihood
analyses we e hen pe o med o each ma ix using
PhyML (Guindon and Gascuel 2003), wi h 1000
eplica es o boo s ap.
Mic osa elli e analyses
Ou o he nine SSR loci, six we e conside ed o be
eliable and we e subsequen ly analyzed. A e com-
pa isons o eplica es, all dubious peaks we e
emo ed. Since mos geno ypes ha e a leas h ee
alleles in his polyploid species (see below), SSR
ma ke s we e analyzed as bina y da a and he ma ix o
allele size was con e ed in o p esence/absence da a
ma ix. Genalex 6.51 so wa e (Peakall and Smouse
2006) was used o sea ch o ma ching geno ypes,
bea ing e idence o iden ical clones. The sea ch o
ma ching geno ype was epea ed accoun ing o one,
wo o h ee allelic e o s. Allelic accumula ion cu es
we e pe o med (specaccum unc ion, egan R pack-
age, Oksanen e al. 2013) o ma ch allelic ichness o S.
e sicolo and S. pa ens.
S uc u e o gene ic di e si y was analyzed using
mul i a ia e analyses o ade4 and adegene packages
o R (D ay and Du ou 2007; Jomba 2008). P incipal
coo dina e analysis (PcoA, dudi.pco unc ion, ade4 R
package) was based on Jacca d dis ances (Jacca d
1901) compu ed on SSR p esence/absence (dis .bina y
unc ion, ade4 R package). Fo ma ching samples
ha ing iden ical geno ypes (clones), only one geno-
ype was kep in analyses based on indi idual geno-
ypes and hese we e iden i ied as ‘‘clones’’ on he
PcoA plo . A disc iminan analysis was conduc ed on
he co ela ion o allele p esence/absence o dis in-
guish gene ic g oups acco ding o he clus e ing
p ocedu e designed by Jomba e al. (2010) (DAPC
analysis, adegene R package). Finally, allele equen-
cies wi hin hese g oups we e used o compu e Nei
dis ances (Nei 1972) and o build a Neighbo Joining
ne wo k o explo e ela ionships be ween DAPC
Spa ina e sicolo Fab e: Ano he case o Spa ina ans-A lan ic in oduc ion? 2127
123
gene ic g oups (nj unc ion, ape R package, Pa adis
e al. 2004).
Resul s
Ch omosome coun s e ealed ha in bo h analyzed
popula ions, S. e sicolo indi iduals ha e 2n =40
ch omosomes (Fig. 1b), indica ing ha his axon is a
e aploid species.
No sequence he e ogenei y was obse ed in ei he
chlo oplas o nuclea (ITS) sequences. In a-genomic
polymo phism migh be expec ed in polyploid nuclea
genomes, bu he ITS egions (belonging o he DNA
gene amily) seem o ha e unde gone conce ed
e olu ion as p e iously ound in o he Spa ina
species (Baumel e al. 2002; Bou e e al. 2015).
Maximum Pa simony (MP) analyses we e pe o med
using he ndhC- nV (601 bp), pe A-psbJ (470 bp),
nL- nF (577 bp), nT- nL (631 bp), chlo oplas
conca ena ed (2278 bp) o nuclea ibosomal ITS
(457 bp) ma ices. These analyses esul ed in 23, 34,
20, 23, 3 and 19 equal mos pa simonious ees. In
hese analyses, S. e sicolo , S. pa ens and S. bake i
always belonged o he same clade (Fig. 2). Fo he
mos esol ed ee co esponding o he ITS egions,
hese h ee species belong o a well-suppo ed clade
(99 %) and a e as posi ioned as a sis e clade o S.
a undincacea and S. densi lo a (100 % boo s ap
suppo ). The sequences ob ained om all S. e si-
colo (including he sequences ob ained by P ie o e al.
2011) and S. pa ens samples we e iden ical, apa om
a single subs i u ion obse ed in one accession o S.
pa ens (Cheesequake s a e Pa k, Flo ida) o he ndhC-
nV egion. S. e sicolo and S. pa ens a e closely
ela ed o S. bake i, p esen ing only wo subs i u ions
(one o he ndhC- nV and one o he ITS egions).
Since simila ee opologies we e ob ained using
Maximum Likelihood, only he MP ees a e p esen ed
he e.
Thi y-se en alleles we e eco ded o e he six SSR
loci. All SSR geno ypes we e he e ozygous wi h
mos ly 3 o 4 alleles pe locus. Wi hin S. e sicolo ,
sea ch o ma ching geno ypes e ealed 35 geno ypes
among 52 samples, 31 being unique and 4 being
epea ed om 4 o 11 imes. These iden ical geno ypes
a e e e ed as ‘‘clones’’. Accoun ing o one, wo o
h ee allele e o s we ound 41, 44 and 50 ma ching
geno ypes in S. e sicolo , whe eas no ma ching
geno ypes we e ound among he 8 samples o S.
pa ens e en accoun ing o 3 allele e o s. Accumu-
la ion cu e (Fig. 3) accoun ing o he unequal
sampling be ween S. e sicolo (n =52) and S.
pa ens (n =8) e ealed highe allelic di e si y in S.
pa ens han in S. e sicolo : i.e. o 6 samples 32
alleles we e encoun e ed in S. pa ens agains 26 in S.
e sicolo (Fig. 3).
The PcoA analysis based on Jacca d dis ances
compu ed be ween indi idual geno ypes (Fig. 4)
e ealed ha he main s uc u e is due o di e en ia ion
be ween 6 ou o 8 S. pa ens geno ypes. The o he 2 S.
pa ens samples a e mo e simila o S. e sicolo
geno ypes. He ba ium specimens a e sca e ed among
S. e sicolo geno ypes, excep one (collec ed in
Ca non, F ance, in 1880 by Jou e) ha is g ouped wi h
S. pa ens (H6, Fig. 4). The he ba ium specimen
collec ed by Fab e in (1849)inAgdehasoneo he
geno ypes eco ded on many indi iduals (‘‘ma ching
geno ypes’’ see Ma e ials and me hods) and iden i ied
as clone ‘‘b’’ (Fig. 4). This clone is ound in F ance,
Basque a ea, I aly and Sicily. Acco ding o he DAPC
analysis (Fig. 4) and NJ ne wo k (Fig. 5), he SSR
geno ypes we e op imally clus e ed in six g oups. The
gene ic clus e numbe 4 ( ep esen ed in g een, Figs. 5,
6) was composed o Eu opean samples om F ance
(na u al popula ions and mos he ba ium samples),
I aly, Co sica, and No h Spain. The indi iduals om
he sou he n Ibe ic Peninsula (sou h o Spain and
Po ugal) and Alge ia a e g ouped in wo (1 and 6)
closely ela ed clus e s. Th ee gene ic clus e s could be
dis inguished in he Ame ican samples o S. pa ens:(1)
Mexico-Flo ida-Delawa e (2) New Je sey and (3) New-
Je sey Hampshi e. The he ba ium sample om F ance
sampled in Ca non (He aul ) was assigned in a g oup
wi h S. pa ens (Mexico-Flo ida-Delawa e g oup).
c
Fig. 2 Molecula phylogeny o Spa ina based on chlo oplas
(andhC- nV: 601 bp; bpe A-psbJ: 470 bp, c nL- nF: 577 bp,
d nT- nL: 631 bp, econca ena ed sequences: 2278 bp) o
nuclea ibosomal ITS sequences (457 bp) using he maximum
pa simony me hod. Fo each phylogeny, one o he equally
pa simonious ees ha is opologically iden ical o he 50 %
majo i y- ule consensus ee (pe A-psbJ: 34 equally pa simonious
ees; conca ena ed chlo oplas sequences: h ee equally pa simo-
nious ees; ITS: 19 equally pa simonious ees) o he 50 %
majo i y- ule consensus ee (ndhC- nV: 23 pa simonious ees;
nL- nF: 20 pa simonious ees) is p esen ed. The boo s ap
pe cen ages (1000 eplica es) a e shown in bold abo e he
b anches and he numbe o changes is indica ed below. The ee is
oo ed using ei he Spo obolus c yp and us o So ghum bicolo
2128 A. Baumel e al.
123
5
S. bake i
S. pa ens (New Je sey, USA)
S. e sicolo (Ba ayo, As u ias, Spain)
S. pec ina a
S. a undinacea
S. densi lo a
S. al e ni lo a
S. ma i ima
Spo obolus c yp and us
S. oliosa
S. pa ens (Tamaulipas, Mexico)
S. pa ens (New Hampshi e, USA)
S. pa ens (Cheesequake s a e, New Je sey, USA)
S. pa ens (Flo ida Deso o Pa k, Flo ida, USA)
S. pa ens (Delawa e, USA)
S. pa ens (Bay iew a enue, New Je sey, USA)
S. e sicolo (Ped ena, Can ab e, Spain)
S. e sicolo (O u , As u ias, Spain)
S. e sicolo (Pan ano, Sicily, I aly)
S. e sicolo (Boudigou de To eilles, Py énées o ien ales, F ance)
S. e sicolo (Kouali, Tipaza, Alge ia)
S. e sicolo (Les A esquie s, Hé aul , F ance)
S. e sicolo (Po Sain Louis, Bouches du Rhône, F ance)
S. e sicolo (E ang des Pesquie s, Va , F ance)
99
70
16
16
6
3
1
21
1
1
7
9
8
2
100
6
93
92
5
F
S. e sicolo (P ie o e al. 2011; JN133292)
S. e sicolo (P ie o e al. 2011; JN133290)
S. e sicolo (P ie o e al. 2011; JN133291)
99
80
4
22
3
3
2
5
27
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
S. bake i
S. pa ens (New Je sey, USA)
S. pec ina a
S. a undinacea
S. densi lo a
S. al e ni lo a
S. ma i ima
So ghum bicolo
S. pa ens (Cheesequake s a e, New Je sey, USA)
S. pa ens (Flo ida Deso o Pa k, Flo ida USA)
S. pa ens (Delawa e, USA)
S. e sicolo (Pan ano, Sicily, I aly)
S. e sicolo (Boudigou de To eilles, Py énées o ien ales, F ance)
S. e sicolo (Kouali, Tipaza, Alge ia)
S. e sicolo (Les A esquie s, Hé aul , F ance)
S. e sciolo (Po Sain Louis, Bouches du Rhône, F ance)
S. e sciolo (Vieux salins, Va , F ance)
S. e sicolo (Guadiana i e , Andalusia, Spain)
S. e sciolo (Banda bianca, Co sica, F ance)
S. e sicolo (Tagus es ua y, Po ugal)
S. e sicolo (G ande mo e, Hé aul , F ance)
S. e sciolo (E ang des Pesquie s, Va , F ance)
S. pa ens (Chissahowi zka, Flo ida, USA)
A
S. bake i
S. pa ens (New Je sey, USA)
S. pec ina a
S. a undinacea
S. densi lo a
S. al e ni lo a
S. ma i ima
Spo obolus c yp and us
S. pa ens (Cheesequake s a e, New Je sey, USA)
S. pa ens (Flo ida Deso o Pa k, Flo ida, USA)
S. pa ens (Delawa e, USA)
S. e sciolo (Bou digou de To eilles, Py énées o ien ales, F ance)
S. e sicolo (Kouali, Tipaza, Alge ia)
S. e sicolo (Les A esquie s, Hé aul , F ance)
S. e sicolo (Po Sain Louis, Bouches du Rhône, F ance)
S. e sicolo (Vieux salins, Va , F ance)
S. e sicolo (Guadiana i e , Andalusia, Spain)
S. e sicolo (Banda bianca, Co sica, F ance)
S. e sicolo (Tagus es ua y, Po ugal)
S. e sicolo (G ande mo e, Hé aul , F ance)
S. e sicolo (E ang des Pesquie s, Va , F ance)
S. pa ens (Chissahowi zka, Flo ida, USA)
S. pa ens (Tamaulipas, Mexico)
S. pa ens (New Hampshi e, USA)
S. pa ens (Bay iew a enue, New Je sey, USA)
S. e sicolo (Ped ena, Can ab e, Spain)
S. e sciolo (O u , As u ias, Spain)
S. e sicolo (Pan ano, Sicily, I aly)
S. e sicolo (Ba ayo, As u ias, Spain)
S. e sicolo (Eb o i e , Andalusia, Spain)
S. e sicolo (Aspe illo dunes, Andalusia, Spain)
S. e sicolo (Roche, Andalusia, Spain)
S. e sicolo (Pan ano, Sicily, I aly)
S. oliosa
1
64
1
1
1 1
1
1
5
3
65
96
B
S. bake i
S. pa ens (Tamaulipas, Mexico)
S. pec ina a
S. al e ni lo a
Spo obolus c yp and us
S. pa ens (New Je sey, USA)
S. pa ens (New Hampshi e,USA)
S. pa ens (Cheesequake s a e, New Je sey, USA)
S. pa ens (Flo ida Deso o Pa k, Flo ida, USA)
S. e sicolo (Aspe illo dunes, Andalusia, Spain)
S. e sicolo (Roche, Andalusia, Spain)
S. e sicolo (Pan ano, Sicily, I aly)
S. e sicolo (Bou digou de To eilles, Py énées o ien ales, F ance)
S. e sicolo (Kouali, Tipaza, Alge ia)
S. e sicolo (Banda bianca, Co sica, F ance)
S. oliosa
S. ma i ima
S. densi lo a
S. g acilis
S. a undinacea
67
3
1
1
2
7
4
3
2
2
3
88
C
S. bake i
S. pec ina a
S. a undinacea
S. densi lo a
S. al e ni lo a
S. ma i ima
Spo obolus c yp and us
S. pa ens (Cheesequake s a e, New Je sey, USA)
S. e sciolo (Bou digou de To eilles, Py énées o ien ales, F ance)
S. e sicolo (Kouali, Tipaza, Alge ia)
S. e sicolo (Les A esquie s, Hé aul , F ance)
S. e sicolo (Po Sain Louis, Bouches du Rhône, F ance)
S. e sicolo (Vieux salins, Va , F ance)
S. e sicolo (Guadiana i e , Andalusia, Spain)
S. e sicolo (Tagus es ua y, Po ugal)
S. e sicolo (G ande mo e, Hé aul , F ance)
S. e sicolo (E ang des Pesquie s, Va , F ance)
S. e sicolo (Ped ena, Can ab e, Spain)
S. e sciolo (O u , As u ias, Spain)
S. e sicolo (Pan ano, Sicily, I aly)
S. e sicolo (Eb o i e , Andalusia, Spain)
S. e sicolo (Aspe illo dunes, Andalusia, Spain)
S. e sicolo (Roche, Andalusia, Spain)
S. e sicolo (Pan ano, Sicily, I aly)
S. oliosa
1
1
1
2
2
1
1
2
4
9
53
91
94
D
S. bake i
S. pa ens (Cheesequake s a e, New-Je sey USA)
S. e sicolo (Bou digou de To eilles, Py énées o ien ales, F ance)
S. pec ina a
S. a undinacea
S. densi lo a
S. al e ni lo a
S. ma i ima
So ghum bicolo
100
85
67
66
54
68
85
12
5
6
9
3
3
3
3
2
1
1
1
0
E
Spa ina e sicolo Fab e: Ano he case o Spa ina ans-A lan ic in oduc ion? 2129
123
The S. pa ens clus e being he mos simila o S.
e sicolo (NJ ne wo k Fig. 6) is obse ed all along
he A lan ic No h Ame ican coas om Mexico o
Delawa e. The clus e 4 which has mos o he
S. e sicolo samples, and he he ba ium sample
collec ed by Fab e, a e he mos simila o S. pa ens
acco ding o he NJ ne wo k (Fig. 6).
Discussion
Ou esul s e eal ha S. e sicolo is a e aploid
species wi h 40 ch omosomes, as ound in S. pa ens
(Ma chan 1968) and no Eu opean o No h-A ican
popula ions analyzed can be di e en ia ed gene ically
om No h Ame ican S. pa ens samples as hey
exhibi simila DNA ITS and cpDNA sequences. In
his monog aph o Spa ina, Mobbe ley (1956) s essed
he mo phological simila i ies be ween hese wo axa,
al hough se e al pheno ypes we e desc ibed o S.
pa ens (Mobbe ley 1956). Ou esul s suppo he
hypo hesis ha all Eu opean and A ican popula ions
o S. e sicolo a e in ac No h Ame ican S. pa ens
in oduced be o e o a he beginning o he nine-
een h-cen u y.
Al hough some mic osa elli e a ia ion was de ec ed
be ween S. pa ens and S. e sicolo , only ew geno ype
di e ences we e obse ed. Gene ic di e ences ega d-
ing mic osa elli e alleles would mos likely esul om
in aspeci ic gene ic di e si y in S. pa ens- e sicolo
popula ions; his is suppo ed by gene ic simila i ies
be ween some No h-Ame ican S. pa ens and S.
e sicolo samples as can be seen on PcoA esul s
(Fig. 4). The in oduc ion o igin could be in he a eas
co e ed by he gene ic clus e 4 (Fig. 5), i.e. F ance,
No h Spain o I aly because he co esponding geno-
ype is he mos simila o S. pa ens in he NJ ne wo k
(Fig. 6). This pa e n is also clea in he PcoA analysis
(Fig. 4). The S. e sicolo samples om Po ugal and
Sou h Spain, o Alge ia a e ei he de i ed om his
in oduc ion o esul ed om a second in oduc ion.
Spa ina pa ens is a highly a iable hizoma ous
species, exhibi ing high ecological ampli ude along
he A lan ic coas o No h Ame ica om Canada o
Cen al Ame ica, colonizing high sal ma sh zones,
beaches and sand dunes, wi h a iable seed se
(Silande and An ono ics 1979). Allozyme s udies in
na i e S. pa ens popula ions (Silande 1984) e ealed
impo an polymo phism wi h a dec eased ole o
ege a i e ep oduc ion om dune o ma sh habi a s.
In con as mic osa elli e geno yping e ealed educed
gene ic di e si y in S. e sicolo compa ed o No h
Ame ican S. pa ens samples (Fig. 3), which is consis-
en wi h a gene ic bo leneck ollowing in oduc ion
in Eu ope oge he wi h he p edominan clonal
p opaga ion o he in oduced plan s. Indeed we ound
17 ma ching SSR geno ypes wi hin S. e sicolo bu
his numbe inc eased o 41 when accoun ing o one
allele e o indica ing ha gene ic a ia ion wi hin S.
e sicolo could be mainly o somaclonal a ia ion.
P elimina y su eys in S. e sicolo popula ions
e ealed s e ile pollen (R. Ami ouche, unpublished
da a), which is in ag eemen wi h he obse a ion ha
his axon a ely p oduces seeds (Fab e 1849; San
Leon e al. 1999; Tison e al. 2014; ou pe sonal
obse a ions). Fu he sampling and phylogeog aphic
analyses a e needed in he na i e egion o S. pa ens o
be e documen he his o y o his axon and o
iden i y he p ecise popula ions ha we e in oduced
in he Medi e anean. Al hough a ious s udies ha e
documen ed dis ibu ion, ecology and plas ici y o S.
pa ens in No h Ame ica (e.g. F asco and Good 1982;
Bu dick and Mendelssohn 1987; Bu dick e al. 1989;
Foo e and Reynolds 1997; Lona d e al. 2010), e y
ew s udies ha e documen ed gene ic di e si y in S.
pa ens (e.g. Wu 2012) and he e is a g ea need o
de elop DNA-based analyses a he genome le el in
he na i e ange o his species, which plays an
impo an ecological ole, p e en ing coas al e osion
and being used in dune es o a ion.
Samples
Alleles
123456
20 25 30 35 40
S. pa ens
Fig. 3 Ra e ac ion cu es o allelic di e si y o he se en SSR
loci
2130 A. Baumel e al.
123