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Pl. Syst. Evol. 264: 217237 (2007) DOI 10.

1007/s00606-006-0511-0 Printed in The Netherlands

Plant Systematics and Evolution

Phylogenetic relationships and genetic diversity of the Salicornieae (Chenopodiaceae) native to the Atlantic coasts of France
E. P. Murakeozy1, A. A nouche2, A. Meudec1, E. Deslandes1, and N. Poupart1
Laboratoire dEcophysiologie et de Biotechnologie des Halophytes et des Algues Marines, (EA 3877 LEBHAM), Institut Universitaire Europeen de la Mer, Universite de Bretagne Occidentale, Plouzane, France 2 Departement dEcologie Evolutive, UMR-CNRS 6553, Universite de Rennes, Rennes Cedex, France Received April 28, 2006; accepted December 7, 2006 Published online: March 6, 2007 Springer-Verlag 2007
1

Abstract. Phylogenetic relationships of members of the tribe Salicornieae, native to the Atlantic coasts of France, were assessed by three molecular markers: the nuclear ribosomal internal transcribed spacer (ITS), the chloroplast trnL-F and the chloroplast matK sequences. In parallel to the phylogenetic studies, a population genetic study was carried out based on randomly amplied polymorphic DNAs (RAPD). Neither the MP/ML analyses of the sequences, nor the AMOVA and NJ analyses of RAPD ngerprints conrmed the morphologybased classication at the specic level within Salicornia. Instead, our investigations are in favour of the species aggregate concept. Two sister groups were revealed in the genus: one is composed of the diploid taxa, while the other clusters the tetraploid taxa. Conicting nuclear versus plastid phylogenetic positions of some tetraploid samples, referred to as S. fragilis, indicate that they most likely derive from a reticulate evolution. Key words: Salicornia, Sarcocornia, glasswort, Chenopodiaceae, phylogeny, ITS, trnL-F, matK, RAPD.

Within the Chenopodiaceae, the tribe Salicornieae Dumort. comprises 13 genera and approximately seventy species, which are dis-

tributed all over the world (Kuhn et al. 1993). Salicornia (c. 15 species) and Sarcocornia (c. 20 species) are among the most diverse genera in the tribe. Salicornia comprises annual species, whereas Sarcocornia groups perennials. Although there is an increasing interest in the production of glassworts (use in the reclamation of salt-aected lands, medicine, alimentation and oil production), the taxonomic treatment of the tribe Salicornieae and, especially, that of Salicornia is still far from satisfactory. The reasons are multiple. The tribe is characterised by highly reduced leaves and owers, and therefore, a simple and similar morphology. Morphological distinction is only possible in fresh state, between owering and fruiting (Gehu et al. 1979). In addition, plants show high levels of phenotypic plasticity (Ingrouille and Pearson 1987, Sagane et al. 2003) and they are mostly cleistogamous (Jeeries and Gottlieb 1982, Noble et al. 1992), although hybridization has been suggested as ` well (Moss 1912, Stace 1997, Lahondere 2004). Based on initial work realised by Ball and Tutin (1959), Gehu et al. (1979) proposed the rst complete classication for the annual

218

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

glassworts of North-Western France, which ` was ultimately revised by Lahondere (2004). Although several authors (e.g. Stace 1997) favour a wider species concept within Salicornia, in which segregates or microspecies, rather than species are distinguished, others (e.g. Gehu and Gehu-Franck 1992, Laho` ndere 2004) prefer a more narrow species view. At the Atlantic coasts of France, ` Lahondere (2004) dierentiate ve diploid species (2n = 18) and three tetraploid ones (2n = 36). The diploid species are named: Salicornia disarticulata Moss (often recognised as S. pusilla J. Woods), S. obscura P.W. Ball & Tutin, S. ramosissima J. Woods, S. brachystachya D. Konig, and S. x marshallii D.H. Dalby (the latter is suggested to be the hybrid of S. disarticulata and S. ramosissima). The tetraploid species are named S. dolichostachya Moss, S. fragilis P.W. Ball & Tutin and S. emerici Duval-Jouve. The taxon name S. brachystachya D. Konig is suggested by Gehu and Gehu-Franck (1992) as an equivalent of S. europaea L., of which the name being ambiguous, should be replaced. However, the Flora of the British Isles recognises the species S. pusilla J. Woods, the hybrid of S. pusilla and S. ramosissima, and two species aggregates: the S. europaea L. aggregate comprising S. ramosissima J. Woods, S. europaea L. and S. obscura P.W. Ball & Tutin; and the S. procumbens Smith aggregate comprising S. nitens P.W. Ball & Tutin, S. fragilis P.W. Ball & Tutin, and S. dolichostachya Moss (Stace, 1997). The genus Sarcocornia is represented by two species in the Atlantic coasts of France; S. fruticosa (L.) A.J. Scott and S. perennis (Miller) A.J. Scott ` (Lahondere 2004). Although their dierentiation seems to be more straightforward, the genus Sarcocornia is not always accepted as distinct from Salicornia (Judd and Ferguson 1999, Freitag 2000) or from Arthrocnemum Moq. (Ball 1993). Table 1 provides a brief overview of the dierent synonyms of the ` above taxa, on the base of Lahondere (2004). Recently, molecular approaches have been applied in order to elucidate the phylogeny of

Salicornioideae using various sets of taxon samplings. Several phylogenetic analyses were carried out within Chenopodiaceae (Kadereit et al. 2003, Schutze et al. 2003, Kadereit et al. 2005) or the subfamily Salicornioideae (Shepherd et al. 2004, Kadereit et al. 2006), but only one study (Papini et al. 2004) focused on infrageneric questions within Salicornia. In addition, population genetic investigations were carried out, with partly contradictory results. An isozyme analysis of Salicornia populations at Stikey (England) conrmed the existence of S. ramosissima and S. europaea sensu stricto (Jeeries and Gottlieb 1982). However, a restriction fragment length polymorphism (RFLP) analysis in the same population demonstrated the existence of habitat-dependent groups, and failed to show any correlation with the previously established two taxa (Noble et al. 1992). Other studies using randomly amplied polymorphic DNAs (RAPD) technique showed correlations between DNA polymorphism and geographical distribution in S. ramosissima (Kruger et al. 2002) and S. europaea (Sagane et al. 2003). The aim of our work was to study the phylogenetic relationships and genetic diversity among members of the tribe Salicornieae using molecular markers. Our rst objective was to test the Salicornia species system of ` Lahondere (2004), as compared to the aggregate system of the Flora of the British Isles (Stace 1997). The Atlantic French coasts nurture a great diversity of Salicornia plants and therefore, seemed particularly suitable for such study. Accordingly, we reconstructed the phylogenetic relationships among the French ` Atlantic species (sensu Lahondere): eight taxa belonging to the genus Salicornia and two to the genus Sarcocornia. Some supposed hybrid specimens, as well as two specimens of A. macrostachyum, originating from the Mediterranean coasts of France (Camargue), were also included. Three molecular markers were applied: the internal transcribed spacer (ITS) of the nuclear ribosomal DNA repeat, the entire trnL-trnF chloroplast DNA region (including the trnL intron and the trnL-F spacer) and a

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

219

Table 1. Species names and synonyms within the Salicornieae native to the Atlantic coasts of France. ` Names are in accordance with Lahondere (2004) with few nomenclatural modications. Symbols indicate ploidy level within Salicornia: ( diploid species; j tetraploid species ` Species name sensu Lahondere Salicornia L. ( S. obscura P.W. Ball et T. G. Tutin Synonyms S. stricta Meyer S. brachystachya Meyer subsp. gracilis S. smithsiana C.E. Moss S. prostrata C.E. Moss S. procumbens Smith S. appressa Dumortier S. europaea L. S. stricta Dumortier S. herbacea L. subvar. brachystachya Meyer S. pusilla C.E. Moss S. pusilla J. Woods S. pusilla J. Woods S. x marshallii J.-M. Gehu S. dolichostachya C.E. Moss subsp. dolichostachya S. procumbens Smith var. procumbens S. stricta Meyer S. procumbens Smith var. stricta Meyer S. oliveri Moss S. lutescens P.W. Ball et T.G. Tutin S. stricta Dumortier S. nitens P.W. Ball et T.G. Tutin S. veneta Pignatti et Lausi S. biennis Alzel S. sarmentosa J. Duval-Jouve S. sarmentosa Gaut S. caespitosa Rouy S. lignose J.Woods Arthrocnemum perenne Moss S. fruticosa L. et auct. S. glauca Stokes Arthrocnemum fruticosum Moq Halocnemum fruticosum Dietr A. fruticosum Moq. var. macrostachyum Moq. S. macrostachya Moric. A. glaucum Ung.-Sternby S. fruticosa L. var. pachystachya Koch. S. glauca Del.

( S. ramosissima J. Woods

( S. brachystachya D. Konig

( S. disarticulata C.E. Moss ( S. x marshallii D.H. Dalby j S. dolichostachya C.E. Moss

j S. fragilis P.W. Ball et T.G. Tutin

j S. emerici J. Duval-Jouve

Sarcocornia A.J. Scott S. perennis (Miller) A.J. Scott

S. fruticosa (L.) A.J. Scott

Arthrocnemum Moquin-Tandon A. macrostachyum (Moric.) K. Koch

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E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts for sequence comparison. In the RAPD study, 21 Salicornia populations were included (Table 2). Plant material was collected as 14 g fresh shoot samples in August, September and October in 2003 and 2004, and stored at 20C until processed. The 2003 sample collection was carried out in cooper` ation with F. Lahondere who helped in species identication. Five local individuals collected in September, 2004 were given provisory identications, with reference to their supposed ploidy level (Salicornia sp. di1, di2, te1, te2 and te3). The sampled populations were chosen at clearly distinct areas, lying about 30 to 300 km from each other (Fig. 1). Frozen plant material and/or extracted DNA for each population used in this study are conserved at the laboratory (N. Poupart). Phylogenetic analyses. DNA analysis. Total DNA content of 100 mg fresh material of

sequence portion of the matK cpDNA region. Second, as the intrageneric relationships of the Salicornia genus remained poorly resolved in the phylogenetic study, we further explored the DNA polymorphism among populations of this genus, using RAPD variation. Materials and methods
Plant material. Single individuals of 27 Salicornia populations corresponding to eight species sensu ` Lahondere (2004) and four Sarcocornia popula` tions of two species sensu Lahondere (2004) from the North-West of France were included in the phylogenetic analyses (Fig. 1, Table 2). In addition, two individuals of Arthrocnemum macrostachyum (Moric.) K. Koch from the Mediterranean region (Camargue, South France) were analysed

Fig. 1. Geographic localisation of the sampling sites of the Salicornieae populations included in this study

Table 2. List of accessions used for the matK, trnL-F and ITS sequencing of the Salicornieae taxa used in this study. The list provides taxon specication, location, population specication, as well as sample identier and GenBank accession number of DNA sequences. Symbol x ` indicates when sample is included in the RAPD ngerprinting. The nomenclature follows Lahondere, 2004. Symbols indicate ploidy level within Salicornia: h diploid species; n tetraploid species Population ITS matK Ria du Conquet Ria du Conquet Ria du Conquet Keremma AY996230 x x x x x AY996231 AY996228 AY996229 AY996253 AY996254 AY996305 AY996304 AY996317 AY996271 AY996270 AY996297 AY996295 AY996293 AY996255 AY996318 AY996294 AY996251 x x x x x x Keremma Saint Goustan Saint Goustan Keremma Saille ramSail 17 ramBatz 34 ramBatz 35 ramBatz 47 ramBatz 50 ramGous 19 ramGous 31 ramGous 40 marConq 20 Saille Saille Batz sur mer Batz sur mer Saint Goustan Saint Goustan Saint Goustan Ria du Conquet disKere 65 disGous 68 disGous 69 ramKere 63 disConq 29 disConq 30 disKere 64 x x disConq 14 AY996227 AY996306 Site description Identication RAPD GenBank accession no. trnL-F AY996268 AY996269

Taxon

Salicornia L. h S. disarticulata Moss

h S. disarticulata Moss h S. disarticulata Moss h S. disarticulata Moss

h h h h

S. S. S. S.

disarticulata Moss disarticulata Moss disarticulata Moss ramosissima J. Woods

h S. ramosissima J. Woods

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

h h h h h h h h

S. S. S. S. S. S. S. S.

ramosissima J. Woods ramosissima J. Woods ramosissima J. Woods ramosissima J. Woods ramosissima J. Woods ramosissima J. Woods ramosissima J. Woods x marshallii D.H. Dalby

AY996252 AY996245

AY996296 AY996288 AY996286 x AY996313 x AY996246 AY996289 marConq 10 marConq 24 marGous 71 marMsar 23

h h h h Ria du Conquet Ria du Conquet Saint Goustan Saint Goustan

S. S. S. S.

x x x x

marshallii marshallii marshallii marshallii

D.H. D.H. D.H. D.H.

Dalby Dalby Dalby Dalby

upper marsh, salty moist pan idem idem upper marsh, sandy soil idem upper marsh idem upper marsh, sandy soil inland hypersaline saltworks idem idem idem idem upper marsh salt pan idem idem upper marsh, salty moist pan idem idem high shore idem

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Table 2. (Continued) Population ITS matK trnL-F Saint Goustan Saint Goustan Le Collet x x x x x x AY996250 AY996316 marColl 73 obsMsar 18 obsColl 74 obsColl 75 obsBell 27 marMsar 41 marMsar 44 marColl 72 x AY996247 AY996314 AY996244 AY996287 Site description Identication RAPD GenBank accession no.

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Taxon

h S. x marshallii D.H. Dalby h S. x marshallii D.H. Dalby h S. x marshallii D.H. Dalby

h S. x marshallii D.H. Dalby

h S. obscura P.W. Ball et T.G. Tutin h S. obscura P.W. Ball et T.G. Tutin

h S. obscura P.W. Ball et T.G. Tutin h S. obscura P.W. Ball et T.G. Tutin

AY996248 AY996315 AY996290

h h h h obsBell 28 obsBell 100 braPlou 58 di1Kere 61 x x

S. obscura P.W. Ball et T.G. Tutin S. obscura P.W. Ball et T.G. Tutin S. brachystachya D. Konig diploid Salicornia sp. di1

AY996249 AY996225 AY996226

AY996292 AY996291 AY996266 AY996267

h diploid Salicornia sp. di2

di2Kere 76

n S. dolichostachya Moss

dolConq 82

AY996234

AY996275

n S. dolichostachya Moss

dolColl 11

AY996232 AY996307 AY996273

n S. dolichostachya Moss Le Collet

idem idem upper marsh, rarely ooded Le Collet upper marsh, rarely ooded Montsarag idem Le Collet muddy lower marsh, channel bank Le Collet idem Bellevue lower limit of shore, muddy, strong tidal inuence Bellevue idem Bellevue idem Aber Ildut sandy middle marsh Keremma upper marsh, sandy soil, no tidal inuence Keremma lower marsh, sandy soil, rarely ooded Ria du Conquet slikke, sandy-muddy lower marsh, strong tide inuence Le Collet slikke, silty lower marsh, strong tidal inuence Le Collet idem dolColl 36 idem dolColl 48 x

AY996272

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

n S. dolichostachya Moss

n S. dolichostachya Moss n S. dolichostachya Moss n S. fragilis P.W. Ball et T.G. Tutin AY996242 AY996285

Le Collet Le Collet Ria du Conquet

dolColl 84 dolColl 85 fraConq 99

x x

AY996233

AY996308

AY996274

n S. fragilis P.W. Ball et T.G. Tutin Le Collet Le Collet Le Collet Le Collet Le Collet Batz sur mer x x x x x AY996235 AY996237 AY996311 Batz sur mer Batz sur mer 2 Bellevue emeBatz 39 emeBat2 88 emeBell 86 fraColl 25 fraColl 26 fraColl 78 fraColl 79 emeBatz 12 x AY996239 AY996240 AY996241 AY996310 fraColl 16 x AY996238

Saint Goustan

fraGous 80

AY996243

AY996312

AY996284 AY996281 AY996280 AY996282 AY996283 AY996277 AY996276 AY996279

n S. fragilis P.W. Ball et T.G. Tutin T.G. T.G. T.G. T.G. Tutin Tutin Tutin Tutin

n n n n n

S. S. S. S. S.

fragilis P.W. Ball fragilis P.W. Ball fragilis P.W. Ball fragilis P.W. Ball emerici J. Duval

et et et et

n S. emerici J. Duval n S. emerici J. Duval n S. emerici J. Duval

n tetraploid Salicornia sp. te1 Le Collet Keremma

Batz sur mer

te1Bat2 66 te2Coll 96 te3Kere 57

x x AY996256

n tetraploid Salicornia sp. te2

n tetraploid Salicornia sp. te3

idem idem limit of slikke and lower marsh, silty channels salty moist pan in middle shore muddy lower marsh, channel bank idem idem idem idem inland hypersaline saltworks idem idem slikke, silty lower salt marsh, strong tidal inuence inland hypersaline saltworks upper shore, rarely ooded silty lower schorre, channel bank sfrMsar 15 sfrMsar 32 sfrMsar 94 speConq 21 x AY996258 AY996259 x x AY996223 AY996320 AY996302 AY996319 AY996300 AY996299 AY996261

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

Sarcocornia A.J. Scott S. fruticosa (L.) A.J. Scott Montsarag Montsarag Montsarag Ria du Conquet

S. fruticosa (L.) A.J. Scott S. fruticosa (L.) A.J. Scott S. perennis (Miller) A.J. Scott

sandy-muddy upper salt marsh idem idem lower schorre

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Table 2. (Continued) Population ITS matK trnL-F Le Collet Keremma upper marsh, sandy soil artCama 52 sfrCama 55 speKere 92 AY996224 silt lower salt marsh speColl 90 Site description Identication RAPD GenBank accession no.

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Taxon

AY996222 AY996301 AY996262 AY996263

Camargue, Sts Maries Camargue, Sts Maries

AY996260 AY996303 AY996265 AY996264

S. perennis (Miller) A.J. Scott S. perennis (Miller) A.J. Scott Arthrocnemum Moq. A. macrostachyum (Moric.) K. Koch A. macrostachyum (Moric.) K. Koch upper marsh, sandy soil upper marsh, sandy soil Location K47 K51 P32 P62 K50 Identication ITS AY489247 AY489251 AY641532 AJ578062 AY489250 matK

Previously published sequences used in this paper Taxon Author Kadereit et al. (2005) Kadereit et al. (2005) Papini et al. (2004) Papini et al. (2004) Kadereit et al. (2005)

trnL-F

Salicornia L. h S. europaea L.

h S. prostrata Pall.

h S. patula Duval-Jouve n S. veneta Pign. et Lausi

n S. procumbens Sm.

SE France : Camargue Romania: Transsylvania,Nires Italy: Foggia Italy: Emilia Romagna, Ravenna Italy: Adria, Belvedere

Sarcocornia A.J. Scott S. fruticosa (L.) A.J. Scott Schutze et al. (2003)

AY181881 AY489257

S. littorea (Moss) A.J. Scott Kadereit et al. (2005)

Kadereit et al. (2005)

SW Spain: Sevilla, Isla S81 Mayor K57 South Africa: Franskraal, Western Cape Australia: Karuah, K59 New South Wales. Spain: Delta del Ebro, K39 Tarragona

AY489259

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

S. quinqueora (Bunge ex Ung.-Sternb. Arthrocnemum Moq. A. macrostachyum (Moric.) K. Koch Kadereit et al. (2005)

AY489239

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

225

A. macrostachyum (Moric.) K. Koch Pachycornia. triandra (F. Muell.) J. Black

Halocnemum strobilaceum Allenrolfea vaginata

individual plants was extracted using DNeasy Plant Mini Kit (QIAGEN, Australia), according to the manufacturers description. The nuclear regions ITS1, 5.8S and ITS2 (hereafter, ITS) were amplied together, using the primer pair ITS5 and ITS4 described by White et al. (1990). An approximately 800 bp part of the chloroplastic matK gene was sequenced using the Amaranthaceae/Chenopodiaceae-specic forward primer ACmatK500F (Hilu et al. 2003) and the reverse primer trnK2R (Johnson and Soltis 1995). The region comprising the trnL intron and the intergenic spacer between the trnL 3 and the trnF (hereafter, trnL-F) was amplied together, using c and f primers of Taberlet et al. (1991). PCR reactions were carried out in 50 ll volumes using an Applied Biosystems GeneAmp 2700 ThermoCycler. Each reaction contained approximately 1020 ng of genomic template DNA, 2 mM MgCl2, 8 pmols of each of forward and reverse primers, 0.2 mM of each dNTP, 1X reaction buer, and 2.5 units DNA polymerase. For amplication of the ITS and matK sequences, PCR conditions were as follows: initial denaturation for 5 min at 95C (ITS) or 94C (matK), 25 thermal cycles of 1 min at 95C (ITS) or 94C (matK), 1 min at 50C and 1 min at 74C, and a nal extension of 5 min at 74C. For the trnL-F amplication the thermal cycling prole consisted of 25 cycles of 1 min at 94C, 1 min at 50C and 2 min at 72C, with an initial denaturation for 5 min at 94C and a nal extension of 5 min at 72C. After thermal cycling, 5ll aliquots of PCR products were checked by electrophoresis on 1.2% agarose gels. PCR products were puried using the GeneClean II. kit (Bio 101, Vista, CA). In few cases, PCR products were puried from a 0,5% TAE agarose gels, using the same purication kit. Puried, double-stranded PCR products were sequenced directly, using the ABI prism Big Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster City, CA) on an ABI 3700 class automated sequencer (PE-Applied Biosystems). PCR primers were used as sequencing primers. Forward and reverse sequences were compared and edited, and consensus sequences initially aligned using Codon Code Aligner (vs. 1.3.4, Li-COR Inc., Dedham, MA). Data analysis. All maximum parsimony (MP) and maximum likelihood (ML) analyses were conducted using PAUP* (Swoord 2001). Gaps

AY489240

K40

K46

AY489246

Kadereit et al. (2005)

Turkey: Adana, Seyhan Kadereit et al. (2005) Australia: south far western plains, New South Wales Muller and Borsch (2005) not specied Muller and Borsch (2005) not specied

AY514842 AY514828

226

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts sequences of Halocnemum strobilaceum and Allenrolfea occidentalis, accession numbers AJ578068 and AY181875, respectively. In addition to the outgroup taxa, the nal ITS data matrix was completed by 11 previously published sequences in order to represent other Salicornieae taxa which are not present in the French Atlantic coasts (listed in Table 2). In the trnL-F analyses Arthrocnemum macrostachyum was used as outgroup. RAPD ngerprinting. DNA amplication. Random amplied polymorphic DNA analysis was carried out only for Salicornia DNA samples. The assays were performed in 12.5ll reaction mixtures containing approximately 50 ng of genomic template DNA, 2 mM MgCl2, 10 pmol of primer, 0.2 mM of each dNTP, 1X reaction buer (Uptima, Interchim, France), and 1.25 unit of Taq DNA polymerase (Uptima, Interchim, France). Primers used for the analysis equalled to primer K01, K15 and M02 used by Kruger et al. (2002) for Salicornia ramosissima J. Woods. Negative controls were included. Thermal cycler (GeneAmp PCR System 2700, Applied Biosystems) program was as described by Kruger et al. (2002). Amplication products were separated by electrophoresis in 2% agarose gels with TAE buer system at 110V for 4 hours. Gels were stained with ethidium bromide, and DNA bands were visualised by UV light and documented using a video camera. Gel pictures were analysed with the software Gene Proler 4.03 (Scanalytics, VA, USA). Data analysis. RAPD bands of all individuals were scored in a data matrix as discrete character states, considering each fragment size as a character present (+) or absent (0). Band intensity was not considered in scoring RAPD bands. All analyses were based on the pair wise squared Euclidian distances among RAPD phenotypes (Excoer et al. 1992). We used AMOVA to estimate the variance components of RAPD patterns, and to partition the variation within and among populations and groups of populations, using Arlequin version 2.000 (Schneider et al. 2000). For each of the 39 individuals, four alternative groupings (geographical, habitat-dependent, taxonomical and ploidy) were tested. In the geographical grouping, populations were assayed according to their locality (4 groups; group 1: Keremma and Ria du Conquet, group 2: Saint Goustan and Montsarag, group 3: Batz sur mer and Saille, group 4: Le Collet and Bellevue.) In the habitat-dependent grouping

were inserted at positions where insertions-deletions (indels) occurred. Separate analyses were performed for each gene region using equally weighted characters, with and without coding the indels (Barriel 1994), and bootstrap value (BV) was calculated. For MP searches, starting trees were obtained using random sequence additions, searched using equally weighted MP (Fitch 1971) with TBR branch-swapping, MULPARS on, Steepest Descent not in eect. Internal support to each node was assessed by bootstrap method with 100 replicates of heuristic search (Felsenstein 1985). ML analyses were carried out using the HKY85 model (Hasegawa et al. 1985) of sequence evolution with the option in which base composition and the transition/transversion (Ti/Tv) parameter were estimated from the data set. Heuristic search methods were used with TBR branch swapping and collapse of zero length branches. Analyses were repeated 100 times with the random addition option. Bootstrap tests were performed using 100 replicates with nearest neighbour interchange (NNI) branch swapping. Parameters for bootstrap tests were xed to values estimated from the MP tree. Congruence between the trnL-F and ITS trees was assessed on a common, 30-sample data set, using PAUP*. Strict topological agreement between the 30-sample strict consensus trees was ascertained by construction of largest common pruned trees (Finden and Gordon 1985). The consensus information index (CI1) of Rohlf (1982) was calculated for each of the 30-sample strict consensus trees and for the combined strict consensus tree. The incongruence index of Mickevich and Farris (1981), IMF, was used to indicate character congruence. Finally, a global test for homogeneity was accomplished by the incongruence length dierence (ILD) test of Farris et al. (1995) on the combined data sets. For this test, invariant characters were excluded (Cunningham 1997), and heuristic searches were conducted using simple addition, TBR branch swapping, MULTREES on. One hundred random repartitions were performed with MAXTREES limit set to 1000. In the phylogenetic analyses, additional sequences obtained from GenBank were used as outgroup: matK sequences of Halocnemum strobilaceum (tribe Salicornieae) and Allenrolfea vaginata (tribe Halopeplideae), accession numbers AY514842 and AY514828, respectively; ITS

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts four habitat types were distinguished: saltwork, lower marsh, upper marsh, salt pan and upper marsh; in the taxonomical assay, populations were grouped according to taxa (11 groups); while in the ploidy grouping diploid and tetraploid samples were assayed. An unrooted neighbour-joining (NJ) dendrogram (Saitou and Nei 1987) was obtained from the matrix using Arlequin v. 2.000 (Schneider et al. 2000), which was implemented in the program Treeview 1.6.6. (Page 1996). A Mantel test (Mantel 1967) was conducted to check whether genetic distances (Reynolds co-ancestry distances; Reynolds et al. 1983) were in correlation with geographical distances. Signicance of the matrix correlation was evaluated by comparing the observed statistics with its random distribution after 1000 permutations using Arlequin version 2.000 (Schneider et al. 2000).

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Results Sequence characteristics and phylogenetic analyses. The nucleotide sequences determined in this study are deposited in GenBank under accession numbers from AY996222 to AY996320 (Table 2). Sequence length and composition of each region sequenced, as well as tree statistics from separate and combined phylogenetic analyses are summarised in Table 3. matK. matK sequences were obtained from 13 Salicornia and four Sarcocornia accessions, and from one accession of Arthrocnemum macrostachyum. The nal matrix consisted of 838 characters, starting about 553 bp in the 3 direction in the coding region. A six bp long insertion was characteristic to the Salicornia genus and was absent from the other examined genera. A similar observation was reported by Pagliano et al. (2005). Pairwise sequence divergence in the non-coded matrix ranged from 0% among species within a genus to 3% among genera. Within the genus Salicornia, diploid and tetraploid taxa only diered by a single nucleotide (0.12% sequence divergence), with the exception of one individual (fraGous 80) from the tetraploid S. fragilis, which had a sequence similar to that of the diploid individuals.

The skewness test indicated the non-random structure of the data matrix (g1 statistic = )0.7428, signicant at P = 0.01). The MP and ML analyses each generated a single tree (Table 3; Fig. 2.a) which shared exactly the same topology, and were only resolved at the generic level. trnL-F. The trnL-F data matrix consisted of 31 Salicornia, ve Sarcocornia and two Arthrocnemum accessions. After end-trimming, the sequences were 719 to 940 nucleotides long, including the tRNA-Leu (trnL) gene and the trnL-F intergenic spacer region. The proper alignment of the sequences required eight indels, of which some are remarkable: a six bp insertion characterizes the genus Salicornia, while another 11 bp long insertion is only shared by members of Sarcocornia. Genetic distances under the HKY85 model ranged from 0.000 to 0.004 within Salicornia; Salicornia and Sarcocornia diered by about 0.05, while the genetic distance was smaller between Salicornia and Arthrocnemum (0.015 to 0.018). The tree length skewness test conrmed that the results were due to non-random structure (g1=)2.1499, signicant at P = 0.01). The MP analysis resulted in two equally parsimonious trees, while the ML search resulted in a single tree (not shown) similar to the MP consensus tree (Table 3; Fig. 2b). They strongly support i) the separate phylogenetic position of Sarcocornia and Arthrocnemum; ii) the monophyly of Sarcocornia (100% BV) without further separation of the two species; and iii) the monophyly of Salicornia (96%). There is a major split in Salicornia between a clade including all the 18 diploid samples plus two tetraploid ones (fraGous80 and fraConq99), and a clade including the remaining 11 tetraploid samples. The monophyly of the tetraploids is signicant (BV 89%), while that of the diploids is less well supported (BV 62%). The tetraploid fraGous80, which clustered within diploids in the matK analysis, also showed trnL-F phylogenetic anities with diploid individuals. ITS. The total length of the entire ITS region (including the 5.8S cistron) ranged from

Table 3. Summary of sequence characteristics and tree statistics for the gene regions used in this study Complete data set matK 12 20 780933 838 3 58 37 32 0.46 1 63 3 4 0.952 0.977 1 1453.80 1 1267.05 1 2338.76 1 1257.53 2 108 2 4 0.991 0.998 12 325 12 10 0.822 0.943 3 65 2 3 1.000 1.000 3 137 3 5 0.985 0.995 2 1401.80 12 203 3 5 0.985 0.995 3 2800.83 719940 786 795 292 60 30 0.98 596820 657 15 204 142 57 1.11 758 33 63 52 645 6 120 70 1432 39 183 70 13 38 23 49 13 30 13 30 13 30 9 12 2271 49 217 133 3 238 5 5 0.987 0.990 1 4127.55 trnL-F ITS trnL-F trnL-F+ ITS matK+trn L-F+ITS ITS Reduced data sets

228

Characteristics

Number of taxa included Number of individuals included Sequence characteristics Length of sequenced region (range) Length of aligned region Number of gaps coded as binary characters Number of variable characters Number of parsimony informative characters Mean GC content (%) Ti/Tv ratio Tree statistics of the parsimony analysis Number of MP trees Tree length No. nodes resolved No. branches with BV80% Consistency index (CI) Retention index (RI) Tree statistics of the ML analysis Number of ML trees -ln L

E. P. Murakeozy et al.: Molecular phylogeny within the French Atlantic glassworts

Ti/Tv ratio: transitions/transversions; MP: most parsimonious; BV: bootstrap value; ML: maximum likelihood; -lnL: log likelihood score

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matK
Hal. strobilaceum All. vaginata Art. mac. Cama 52

trnL-F
Art. mac. Cama 55 Art. mac. Cama 52 Sar. perennis Conq 21 Sar.perennis Kere 92 Sar. perennis Coll 90 Sar. fruticosa Msar 32 Sar. fruticosa Msar 15 S.emerici Bell 86 S.emerici Batz 12 S.emerici Batz 39 S.fragilis Coll 78 S.fragilis Coll 26 S.fragilis Coll 16 S.fragilis Coll 25 S.dolichostachya Coll 36 S.dolichostachya Conq 82 S.dolichostachya Coll 84 S.dolichostachya Coll 11 Salicornia sp. Kere 61 S.brachystachya Plou 58 S.disarticulata Gous 69 S.disarticulata Kere 65 S.disarticulata Conq 29 S.disarticulata Conq 14 S.obscura Bell 28 S.obscura Bell 100 S.obscura Bell 27 S.ramosissima Kere 63 S.ramosissima Gous 40 S.ramosissima Sail 17 S.ramosissima Batz 47 S.ramosissima Batz 34 S.marshallii Conq 20 S.marshallii Coll 72 S.marshallii Msar 23 S.marshallii Conq 10 S.fragilis Gous 80 S.fragilis Conq 99

100 70

67 100 100

Sar. fruticosa Msar 15 Sar. fruticosa Msar 94 Sar. perennis Leco 21 Sar. perennis Coll 90 S.fragilis Gous 80

100

89

100

71

S.dolichostachya Coll 84 S.dolichostachya Coll 11 S.emerici Batz 12 S.emerici Bell 86 S.disarticulata Kere 65 S.disarticulata Conq 29 S.x marshallii Gous 71 S.x marshallii Msar 44 S.obscura Bell 27 S.obscura Coll 74 S.ramosissima Sail 35 S.ramosissima Kere 63

100

100

62

ITS

100 99 100 56 100 98 61

51 57 68

80 53 87

100 Salicornia

99

85 66

Hal.strobilaceum P68 All.occidentalis S75 Art. mac. Cama 52 Art.mac.K39 Art.mac. K40 Pac.triandra K46 Sar.fruticosa S81 Sar.fruticosa Msar 15 Sar.fruticosa Msar 32 Sar.perennis Conq 21 Sar.perennis Coll 90 Sar.perennis Kere 92 Sar.littorea K57 Sar.quinqueflora K59 S.procumbens K50 S.veneta P62 Salicornia sp. Kere 57 S.emerici Batz 39 S.emerici Bell 86 S.fragilis Gous 80 S.fragilis Coll 78 S.fragilis Coll 26 S.fragilis Coll 25 S.fragilis Coll 16 S.fragilis Conq 99 S.dolichostachya Conq 82 S.dolichostachya Coll 11 S.dolichostachya Coll 84 S.europaea K47 Salicornia sp. Kere 61 S.x marshallii Coll 72 S.brachystachya Plou 58 S.obscura Bell 100 S.obscura Bell 27 S.ramosissima Gous 40 S.ramosissima Gous 19 S.ramosissima Sail 35 S.ramosissima Sail 17 S.disarticulata Gous 69 S.disarticulata Conq 14 S.disarticulata Kere 65 S.disarticulata Kere 64 S.patula P32 S.prostrata K51 S.ramosissima Kere 63 S.obscura Msar 18 S.x marshallii Conq 20 S.x marshallii Msar 44 S.x marshallii Msar 23

Combined trnL-F+ITS

100 93 63

70 90

100

100

Art. mac. Cama 52 Sar. fruticosa Msar 15 Sar. fruticosa Msar 32 Sar. perennis Coll 90 Sar. perennis Conq 21 Sar. perennis Kere 92 S. fragilis Conq 99 S. fragilis Gous 80 S. dolichostachya Coll 84 S. dolichostachya Conq 82 S. dolichostachya Coll 11 S. fragilis Coll 78 S. fragilis Coll 26 S. fragilis Coll 25 S. fragilis Coll 16 S. emerici Batz 39 S. emerici Bell 86 S. disarticulata Conq 14 S. disarticulata Kere 65 S. disarticulata Gousv 9 S. obscura Bell 100 S. obscura Bell 27 S. ramosissima Kere 63 S. ramosissima Gous 40 S. ramosissima Sail 17 S.x marshallii Coll 72 S.x marshallii Msar 23 S.x marshallii Conq 20 S. brachystachya Plou 58 Salicornia sp. Kere 61

Fig. 2. Molecular phylogenetic analyses of the French Atlantic Salicornieae. Presented are the strict consensus trees inferred from MP analyses of the (a) matK, (b) trnL-F, and (c) ITS regions, and the consensus tree derived from the analysis of the combined trnL-F and ITS data (d). Indels were coded, bootstrap condence values above 50% (100 replicates) are above the branches. Tetraploid Salicornia samples are marked with grey boxing. Vertical bars alongside the tree indicate: solid: Salicornia, dotted: Sarcocornia; double: other genera

625 to 626 bp in the surveyed 37 accessions (31 Salicornia, six Sarcocornia, one Arthrocnemum). No evidence of obvious ITS variants

was detected within the examined accessions. The ITS1 fragment (239-240 bp) is proved to be more length-variable than the ITS2 (221 bp)

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and provided more phylogenetically informative sites (38 and 32, respectively). Genetic distances in the non-coded matrix containing the French Atlantic samples ranged from 0 to 0.019 within Salicornia, and from 0.003 to 0.110 in Sarcocornia and Arthrocnemum. In order to gain more information on the phylogeny of the Salicornieae, we included various sequences from Genbank to represent other taxa that do not occur along the French Atlantic coasts (see Table 2). The nal matrix contained 49 sequences of 23 taxa. The skewness test conrmed the non-random structure of the data set (g1=)0.7921, significant at P = 0.01). The MP analysis of the ITS sequences yielded 12 MP trees. The ML search resulted in a single tree (not shown), which showed essentially the same branching pattern as found in the strict consensus of the MP trees (Table 3; Fig. 2c). These data strongly support Salicornia as a monophyletic group (BV 100%) and the diploid and tetraploid Salicornia taxa as separate clades (with 99% and 80% BV, respectively). Relationships within these groups remain largely unresolved. The few sequence dierences among the dierent diploid Salicornia accessions do not correlate with the taxonomy or with the geographical origin of the samples. However, two small diploid subclades are revealed: one supports a close relationship between S. patula and S. prostrata (BV = 85%); the other poorly supported (66%) subclade contains some individuals of S. x marshallii, one individual of S. obscura and one of S. ramosissima. Intriguingly, all other samples of the latter taxa fall within the unresolved diploid polytomy. Within Sarcocornia, the European samples of S. fruticosa and S. perennis form a separate clade (100% BV) from the South African or Australian samples (S. littorea K47 and S. quinqueora K59). S. perennis and S. fruticosa appear as two divergent entities, although only moderately supported (57% and 68% BV, respectively). Finally, S. perennis appears to derive from within S. fruticosa, which then appears as a paraphyletic taxon.

Comparison of the three DNA regions. The most conspicuous dierence found between the ITS and the plastid DNA-based topologies is the conicting position of two S. fragilis samples, which are alternatively placed within the tetraploid or the diploid clade, respectively. The other observed dierences clearly result from more resolution in the trnL-F phylogeny relative to the matK, and the ITS phylogeny relative to the plastid DNA regions (see Fig. 2ac). The combined analysis of the ITS and trnL-F data sets with the 30 common individuals reveals four largest common pruned trees (not presented). The construction of these trees required removal of three samples, which were: disGous 69, fraGous 80 or fraConq 99 and sfrMsar 15 or sfrMsar 32, for the four agreement subtrees, respectively. The Rohlfs index for the trnL-F and ITS consensus trees are 0.282 and 0.313, respectively, while for the combined consensus tree it is 0.361, indicating a topological incongruence (CI1<1.0) between the two trees. Character congruence between the trnL-F and ITS data sets was assessed by the Mickevich and Farris index. The IMF value indicates that 21% of the total character incongruence is attributable to dierences between the trnL-F and the ITS data. The incongruence length dierence (ILD) test shows a lack of homogeneity between the two 30-sample data sets (P=0.224). The combined tree (Fig. 2d) reinforces the main conclusions of the two separate analyses: i) Salicornia is well-supported as a monophyletic group, distinct from Sarcocornia; ii) the few representatives of Sarcocornia included here (S. fruticosa and S. perennis) form a monophyletic group iii) two distinct sister subclades are circumscribed in Salicornia, one containing the diploid samples and one grouping the tetraploid samples. The above conclusions are further supported when the three data sets (matK+trnLF+ITS) are analysed together (Table 3). RAPD analysis. The RAPD analysis of 39 Salicornia plants from 21 populations with three dierent primers yielded 99 polymorphic bands (primer K01: 32 bands, primer K15: 28 bands, primer M02: 39 bands) which allowed

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us to distinguish 39 dierent RAPD phenotypes. The AMOVA data extracted from the Euclidean distance matrix was used to test the level of signicance of the partition of genetic variance resulting from dierent groupings of populations (Table 4). Whereas a geographical arrangement of populations does not result a signicant Uct (P>0.1), the habitat-dependent (Uct=0.118; P=0.016), the taxonomical (Uct=0.267; P<0.001) or the ploidy (Uct=0.335; P<0.001) grouping leads to signicant Uct values. Among the three arrangements, the taxonomic and the ploidy grouping yields by far the highest Uct values. However, in all cases, most of the total molecular variance is attributable to divergence among individuals (61 to 73%). The relationships among the 39 individuals is represented by a dendrogram (Fig. 3) constructed by using the Euclidian distance matrix and the NJ method. The branch lengths of the unrooted dendrogram indicate a genetic separation between diploid and tetraploid Salicornia species. Tetraploid populations appear as derived from diploid ones. The molecular variance is smaller within the tetra-

ploid than within the diploid group. Compared to the above, only minor dierences were found when data produced by each of the three primers were analysed separately (data not shown). The Mantel test conducted to analyse isolation by distance, using pairwise Ust values obtained by AMOVA, revealed no correlation between geographical and genetic distances (r = 0.014; P = 0.620). Discussion Generic relations. All phylogenetic analyses, reconstructed here from both plastid (matK and trnL-F) and nuclear (ITS) sequences, clearly distinguished the members of the Salicornia, Sarcocornia and Arthrocnemum genera (Fig. 2 ad). This result is in agreement with most relevant molecular studies (Kadereit et al. 2005, Shepherd et al. 2004, Luque et al. 1995), except with that of Papini et al. (2004) who suggested to include Sarcocornia within Arthrocnemum; a conclusion most probably due to a limited sampling and/or sample misidentication. A further support to our view is provided by the parallel and independent work of Kadereit et al. (2006). The latter,

Table 4. Results of analysis of molecular variance (AMOVA) for 21 Salicornia populations under four alternative groupings (geographical, habitat-dependent, taxonomical and ploidy) d.f. Geographical Among sites Among populations Within populations Habitat-dependent Among habitats Among populations Within populations Taxonomical Among species Among populations Within populations Ploidy Among species Among populations Within populations 3 17 18 3 17 18 10 10 18 1 19 18 Variance )2.21 4.26 11.111 1.039 3.266 11.111 4.131 0.243 11.111 6.148 1.085 11.111 % Total )1.46 28.12 73.34 6.74 21.19 72.08 26.68 1.57 71.75 33.52 5.91 60.57 U Statistics )0.014 0.277 0.267 0.067 0.227 0.279 0.267 0.021 0.282 0.335 0.089 0.394 P 0.643 0.002 <0.001 0.096 0.006 <0.001 <0.001 0.644 <0.001 <0.001 0.045 <0.001

within sites

within habitats

within species

within species

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S. disarticulata Conq 29 S. disarticulata Conq 30 S. ramosissima Batz 35 S.x marshallii Msar 41 S. disarticulata Gous 68 Salicornia sp. Kere 61 S.x marshallii Msar 23 S.x marshallii Coll 73 S.x marshallii Coll 72 S. ramosissima Gous 19 S. ramosissima Batz 47 S. disarticulata Kere 65 S. disarticulata Gous 69 S.x marshallii Conq 20 S. ramosissima Kere 63 S. obscura Coll 75 S. ramosissima Batz 50 S. obscura Bell 28 S. ramosissima Sail 17 S. ramosissima Gous 31 S. dolichostachya Coll 48 S. dolichostachya Coll 11 S. emerici Bat2 88 S. emerici Bat2 89 S. emerici Batz 39 S. dolichostachya Coll 85 Salicornia sp. Coll 96 Salicornia sp. Bat2 S. fragilis Coll 79 S. fragilis Coll 25 S. fragilis Coll 16 S. fragilis Coll 78 S. obscura Msar 18 S.x marshallii Conq 24 S. obscura Bell 27 S. obscura Coll 74 S. emerici Batz 12 S. dolichostachya Coll 84

Salicornia sp. Kere 76 10

Fig. 3. Unrooted Neighbour-joining dendrogram of 39 populations representing 12 Salicornia taxa (eight ` species sensu C. Lahondere) natives to the Atlantic coasts of France, generated from the Euclidian distance matrix. Population and taxon names are as in Table 2. Tetraploid Salicornia samples are marked with grey boxing

ITS- and atpB/rbcL-based study, which provides a more representative generic sampling, conrms that Arthrocnemum is sister to the Salicornia/Sarcocornia clade. Sarcocornia. Although both plastid markers support a common ancestor to all members of Sarcocornia (Fig. 2 ab) with a remarkable 11 bp synapomorphous insertion in the trnL-F region, the inclusion of additional sequences representing South African and Australian Sarcocornia taxa in the ITS phylogeny does not conrm its monophyly; instead, Sarcocornia appears as paraphyletic to Salicornia. This result is highly congruent with the work of Kadereit et al. (2006), who suggest that the infrageneric relationships within the Sarcocornia/Salicornia clade need to be revisited. In the ITS analysis, S. fruticosa appears as paraphyletic to S. perennis (Fig. 2c). In fact, the S. fruticosa S81 sample characterised by Schutze et al. (2003) originates from the Mediterranean coasts of Spain, while our two samples were collected at the Atlantic French coasts. Whether the observed genetic pattern is

in relation with the geographic origin, as indicated for the genus Salicornia (see below), with interspecic hybridisation, as described by Figuera et al. (2003) between S. fruticosa and S. perennis, or, with other reasons [e.g. Shepherd and Yan (2003) suspects a so far unrecognised Spanish taxon] needs further investigations. Salicornia: phylogenetic relations. The genus Salicornia is clearly assessed as a monophyletic group (Fig. 2 ad). The common origin of the Salicornia members is enhanced by two shared indels of 6 bp each in the matK and the trnL-F regions. With the exception of two S. fragilis individuals, the tetraploid taxa are clearly isolated in a monophyletic group, and are placed as sister group to a distinct, moderately to well-supported clade, containing all diploids (Fig. 2 ad). None of the genetic markers used in this study allowed circumscription of the Salicornia species determined in many previous taxonomic studies. No phylogenetic evidence is found that supports S. disarticulata (syn. S. pusilla) as a distinct

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entity. No evidence of the supposed hybrid origin of S. x marshallii is provided either. However, morphologically both S. disarticulata and S. x marshallii are clearly distinguished. In opposition with the general three orets per cyme present in most Salicornia taxa, S. disarticulata has one single oret per cyme, while S. x marshallii is characterised by one, two or three orets per cyme and has so been proposed as hybrid between S. disarticulata and S. ramosissima (Dalby 1975). Other characters (distribution, colour, size and the length of the owering spike) also place S. x marshallii as an intermediate between S. disarticulata and S. ramosissima. Moreover, in the eld, populations of S. x marshallii are often surrounded by populations of S. ramosissima and S. disarticulata. Intriguingly, ITS sequences of dierent populations of S. x marshallii (3 individuals) and S. ramosissima (1) share one synapomorphous nucleotide change (see subclade in Fig. 2c), which could mean that these populations derive from a common ancestor; but this remains poorly supported. One sample of S. obscura, which is also suggested to form hybrids ` with S. ramosissima (Lahondere 2004), also belongs to the same subclade. Nevertheless, other individuals of the former three taxa fall within the wide unresolved diploid polytomy. Considerations on the origin of polyploids in Salicornia. The above results allow the following suggestions: (1) Initially, Salicornia split into two main sister lineages, which arose from a common diploid ancestor about 1.8-1.4 Mya ago; according to the age estimation of Kadereit et al. (2006). (2) One lineage appears to have generated almost all diploid taxa through a relatively recent and rapid diversication process, as it may be illustrated by the lack or the very low sequence divergence among taxa, contrasting with the broad geographical distribution and the striking morphological heterogeneity. (3) The very high ITS sequence identity exhibited among the tetraploid samples is suggestive of a recent and rapid polyploidization process. As no extant diploid representative has been found in this clade, despite a fairly large sampling, it might

be hypothesised that the polyploids have inherited and retained their ITS nuclear rDNA sequences from a common diploid progenitor, which is closely related but distinct from the extant members of the diploid lineage, and which now might have disappeared. (4) Also, it is obvious from the plastid data that almost all polyploids inherited their cytoplasmic genome from the same ancestor. The presumably extinct diploid ancestor would have been rapidly replaced by polyploids, which often exhibit a broadest genetic and ecological potential (e.g. Schierenbeck and A nouche 2005). Two tetraploid individuals among the six ones sampled from dierent localities under the name of S. fragilis (fraGous 80 and fraConq 99) were positioned as members of the tetraploid clade based on nuclear rDNA sequences, and as members of the diploid clade based upon plastid sequences. Such hard incongruence (sensu Wendel and Doyle 1998) between a nuclear (biparentally inherited) and a chloroplastic (maternally inherited) DNA region most likely results from a hybridisation or allopolyploidisation process. In this particular case, the data suggest an allopolyploid mode of formation, which involved individuals from the unknown diploid ancestor of the tetraploid lineage, as paternal progenitors, and individuals from the diploid lineage, as maternal progenitors (providing the plastid genome). It may be hypothesised from the present data that the absence of the ITS type of the maternal parent in the nuclear genome of these individuals might be due to sequence homogenisation towards the paternal type via active processes of interlocus concerted evolution and/or introgression (Wendel et al. 1995). It has been shown that in certain cases such processes may operate very rapidly in the rst generations following the hybrid formation (Fuertes Aguilar et al. 1999). However, this hypothesis should be tested either through a broader sampling of the intra-genomic ITS diversity, and/or through the use of other single-copy or low-copy nuclear genes.

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Salicornia: geographic considerations. Within both the diploid and the tetraploid group, geographic subgroups can be dierentiated. In the ITS analysis two diploid Salicornia samples originating from Italy (S. patula) and Romania (S. prostrata) constitute a monophyletic geographical subgroup that is distinct from the French Atlantic diploid samples. Also, the Atlantic tetraploid populations (monophyletic group with 87% bootstrap) are dierentiated from the two Italian tetraploid samples (S. procumbens K50 and S. veneta P62). A similar geographic distinction is found in an ETS-based study by Teege and Kadereit (2005). The latter authors suggest a possible Mediterranean/inland versus Atlantic grouping within Salicornia. In a nuclear rDNA analysis (Noble et al. 1992) including both diploid and tetraploid Salicornia samples, representatives of Spanish and Saudi-Arabian populations appeared surprisingly uniform and dierent from the British populations. Therefore, one can hypothesise that Salicornia plants invaded the Mediterranean and the Atlantic coastal habitats through two independent colonisation events. However, in our analysis, the S. europaea K47 sample, which, according to the sample description, originates from the Mediterranean French coasts, appeared in the unresolved diploid polytomy, near the French Atlantic samples. It would be interesting to examine other samples from the same population to rule out whether it is an artefact or an interesting addition to the understanding of the Salicornia evolutionary history. Population study. As a complementary approach to the molecular phylogenetic analyses, the variability of RAPD markers has been examined at the population level. The combination of polymorphic bands with the three primers allowed us to distinguish all the 39 sampled individuals. This high genetic variation is surprising in view of the highly inbreeding nature of Salicornia plants. However, as the actual population sizes were not measured and only one or two samples represented each population, the contribution of

this factor to the observed RAPD pattern cannot be ruled out at present. The NJ dendrogram based on pairwise co-ancestry distances showed a high level of intraspecic polymorphism. It could not be related to the geographical situation of the populations (neither in the AMOVA, nor in the Mantel test) and only to a limited extent to their position on the salt marsh. At least two contrasting evidences are known within the Salicornia genus. Sagane et al. (2003) reports that genetic dierences between populations of S. europaea L. occurring at three sites in Hokkaido and Okayama, in Japan, can be well explained on the base of geographic distribution in these populations. Also, Kruger et al. (2002) found some correlation between genetic dierences of S. ramosissima populations and their geographic distribution in Central Germany. In our study the distribution of genetic polymorphism showed the highest Uct values in the taxonomical and in the ploidy grouping of individuals, showing that individuals belonging to either ploidy level are genetically very homogeneous. In addition, as already shown by the sequence data, the level of similarity is higher within the tetraploid group. The latter can be explained by a more recent evolutionary history (see above) and the higher inclination for outbreeding of the tetraploid taxa. The multiple origin of the tetraploid group is indirectly indicated by the fact that in general terms, populations of autopolyploids are expected to maintain higher levels of heterozygosity than do their diploid progenitors (Soltis and Soltis 2000). Concluding remarks. The overall picture arising from the present study makes evident the great genetic homogeneity of members of the genus Salicornia in the Atlantic coasts of France. Although the diploid and tetraploid Salicornia taxa are clearly separated, the genetic diversity is very low within groups of the same karyotype. This provides support to the species aggregates concept proposed by Stace (1997), with however the inclusion of S. disarticulata and S. x marshallii in the S. europaea L. aggregate. It is interesting to

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note that within the diploid and tetraploid groups a broad-term geographic subdivision (Atlantic European and Mediterranean/ inland European) has been observed. The present work strongly suggests that the high morphological variability within the Salicornia genus results more from phenotypic plasticity than from species divergence, but this statement needs to be further assessed on the base of comparative analyses of both natural populations and their cultivated progenies in homogeneous controlled experimental conditions.
We would like to thank the Conseil General du ` Finistere and L. Erdei for supporting E.P. Murakeozy with a post-doctoral fellowship. We are ` very grateful to C. Lahondere, J. Le Bail and F. Bioret for their help in species identication, and to G. Charrier for useful discussions on RAPD analysis and data evaluation. Contribution No. 1021 of the IUEM, European Institute for Marine Studies (Brest, France).

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Addresses of the authors: Eva Patricia Murakeozy (correspondence; e-mail: e.murakeozy@ vodafone.nl), Anna Meudec (e-mail: Anna.Meudec @univ-brest.fr), Eric Deslandes (e-mail: Eric. Deslandes@univ-brest.fr), and Nathalie Poupart (correspondence, e-mail: Nathalie.Poupart@univbrest.fr), Laboratoire des Halophytes et des Algues Marines, (EA 3877 LEBHAM), Institut Universi taire Europeen de la Mer, Universite de Bretagne Occidentale, Technopole Brest-Iroise, 29280 Plouzane, France. Abdelkader A nouche (e-mail: abdelkader.ainouche@univ-rennes1.fr), Research Group Populations and Species Evolutions, UMR 6553 CNRS-University of Rennes 1. Campus Universitaire de Beaulieu, 35042 Rennes Cedex, France.

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