Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction?

HAL Id: hal-01355664 https://hal.science/hal-01355664 Submitted on 25 Apr 2018 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction? Alex Baumel, Mathieu Rousseau-Gueutin, C. Sapienza-Bianchi, Agnès Gareil, N. Duong, Hélène Rousseau, Olivier Coriton, Rachid Amirouche, S. Sciandrello, B. Duarte, et al. To cite this version: Alex Baumel, Mathieu Rousseau-Gueutin, C. Sapienza-Bianchi, Agnès Gareil, N. Duong, et al.. Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction?. Biological Invasions, 2016, 18 (8), pp.2123-2135. �10.1007/s10530-016-1128-z�. �hal-01355664� INVASIVE SPARTINA Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction? A. Baumel .M. Rousseau-Gueutin .C. Sapienza-Bianchi .A. Gareil .N. Duong . H. Rousseau .O. Coriton .R. Amirouche .S. Sciandrello .B. Duarte .I. Cac¸ador . J. M. Castillo .M. Ainouche Received: 23 March 2015 / Accepted: 20 March 2016 ÓSpringer International Publishing Switzerland 2016 Abstract Intercontinental introductions are widespread in the genus Spartina, with important ecological and evolutionary consequences. The native or introduced status of Spartina species is then critical with regard to biodiversity assessment, especially for vulnerable Mediterranean coastline ecosystems. Spartina versicolor was first recorded in southern France in 1849, then successively in various places on the European and North-African Mediterranean and Atlantic coasts. This species is considered to be either a European native or an invasive species introduced from North America which has a high morphological similarity to the Atlantic American species Spartina patens. We performed extensive sampling of S. versicolor in Europe and North Africa (from natural populations and herbarium collections) and compared these samples to other European and American Spartina species (including S. patens). Chromosome counts were reported for the first time and revealed that S. versicolor is tetraploid (2n=4x=40). Phylogenetic analyses based on chloroplast and nuclear ribosomal DNA sequences did not reveal any molecular variation within S. versicolor. In this species, a single haplotype, that is identical to one haplotype of S. patens, was found in the four chloroplast and the nuclear ribosomal ITS regions investigated. In addition, simple sequence repeat markers were used and revealed a low level of genetic diversity within S. versicolor, suggesting that the introduction of S. Guest editors: Alan Gray and Malika Ainouche/Invasive Spartina. Electronic supplementary material The online version of this article (doi:10.1007/s10530-016-1128-z) contains supplementary material, which is available to authorized users. A. Baumel N. Duong Aix Marseille Universite ´, Institut Me ´diterrane ´en de Biodiversite ´et d’Ecologie (IMBE, UMR CNRS, IRD, Avignon Universite ´), Technopo ˆle de l’Environnement Arbois-Me ´diterrane ´e, BP 80, 13545 Aix-en-Provence Cedex 04, France M. Rousseau-Gueutin C. Sapienza-Bianchi  A. Gareil H. Rousseau M. Ainouche (&) UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l’Univers de Rennes), Universite ´de Rennes 1/Universite ´Europe ´enne de Bretagne, 35042 Rennes, France e-mail: [email protected] M. Rousseau-Gueutin INRA, UMR 1349, Institut de Ge ´ne ´tique, Environnement et Protection des Plantes, 35653 Le Rheu Cedex, France O. Coriton Plate-Forme de Cytoge ´ne ´tique Mole ´culaire, INRA, UMR 1349, Institut de Ge ´ne ´tique, Environnement et Protection des Plantes, 35653 Le Rheu Cedex, France R. Amirouche Universite ´des Sciences et de la Technologie Houari Boumediene, Laboratoire de Biologie et Physiologie des Organismes, BP 32 El-Alia, 16111 Bab-Ezzouar, Alger, Algeria 123 Biol Invasions (2016) 18:2123–2135 DOI 10.1007/s10530-016-1128-z versicolor occurred from a narrow genetic pool of S. patens from North America. Keywords Cordgrass Genetic diversity Species status Mediterranean Microsatellites Phylogeny Introduction Wetland habitats are among the most threatened in the Mediterranean as a consequence of intense urbanization, anthropogenic disturbance and increased number of invasive taxa (Me ´dail and Verlaque 1997). In France, Mediterranean wetlands are among the habitats that are the most colonized by invasive species (Verlaque et al. 2002). Because surveys of biodiversity are generally poorly coordinated in the Mediterranean biodiversity hotspot (Marignani et al. 2014), inference of the native or introduced plant species status is not trivial. This status is an essential parameter for biodiversity management and conservation biology. It also represents critical information with regard to population and species evolutionary history. Establishing native status for a species in a given region is not an easy task and requires a combination of different approaches to elucidate the origin, mode of formation and biogeography of the considered taxon. The increased opportunities for long-distance humanmediated species dispersal make these researches even more complex (Kowarik 2003). In this context, molecular markers and evolutionary genetics provide important insight to trace back population, species origin and migration history (Mansion et al. 2008; Hardion et al. 2014). In Spartina (cordgrasses), recurrent intercontinental introduction events and biological invasions are particularly common and well-documented (Daehler and Strong 1996a,b;SanLe ´on et al. 1999;Baumeletal. 2001;Sanche ´z-Gullon 2001; Ayres et al. 2004;Anetal. 2007;Ainoucheetal.2009; Campos et al. 2004; Lonard et al. 2010; Saarela 2012; Strong and Ayres 2013). This grass genus (Poaceae, Chloridoideae) represents a wellsupported monophyletic lineage (Baumel et al. 2002; Fortune et al. 2007) closely related to some members of the paraphyletic Sporobolus genus and Calamovilfa (Peterson et al. 2014). It is composed of about 15 perennial species that have diversified mostly in the New World (Mobberley 1956). Introduction of species outside their native range over the past 150 years has accelerated diversification by facilitating hybridization with native species, introgression or speciation, resulting in several superimposed divergent genomes that coexist in the species currently found in the wild (Ainouche et al. 2012). The basic (haploid) chromosome number in Spartina is considered to be x=10 (Marchant 1968), and all species recorded to date are polyploid, ranging from tetraploids to dodecaploids. Molecular phylogenies from nuclear and chloroplast DNA sequences have indicated that genus Spartina has evolved through two main lineages including tetraploid and hexaploid species respectively (Baumel et al. 2002). The tetraploid lineage is composed of species native to the New World, colonising coastal or inland salt marshes from either Northern (Spartina patens,Spartina bakeri, Spartina gracilis,Spartina cynusoroides, Spartina pectinata)orSouthern(Spartina ciliata, Spartina arundinacea) hemispheres. The tetraploid S. argentinensis (syn. S. spartinae), which has a disjunct distribution in North-Central America and in SouthAmerica, is sister to the hexaploid lineage. This later clade is composed of Spartina maritima,Spartina alterniflora,andSpartina foliosa, all colonizing low marsh zones. Spartina maritima, native to the Western Europe and African Atlantic coasts, isone ofthe few Old World native species with recent taxa of hybrid origin and the controversial S. versicolor (see below). Accidental or deliberate introductions lead to various hybridization events within or between the tetraploid and hexaploid lineages (reviewed in Ainouche et al. 2012; Strong and Ayres 2013). In Europe, introductions of the hexaploid S. alterniflora, native to the Atlantic American coasts and its subsequent hybridization with hexaploid S. S. Sciandrello Department of Biological, Geological and Environmental Sciences, University of Catania, via Alongo 18, 95125 Catania, Italy B. Duarte I. Cac¸ador MARE – Marine and Environmental Sciences Centre, Faculty of Sciences of the University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal J. M. Castillo Departamento de Biologı ´a Vegetal y Ecologı ´a, Facultad de Biologia, Universidad de Sevilla, Apartado 1095, 41080 Seville, Spain 2124 A. Baumel et al. 123 maritima led to the formation of two sterile F1 hybrids in Southern England (S. x townsendii) and in Southwest France (S. x neyrautii). Genome duplication in the British hybrid resulted in the vigorous and fertile allododecaploid S. anglica (Hubbard 1968; Gue ´ne ´gou et al. 1988; Gray et al. 1990; Gray et al. 1991). This species rapidly expanded in range and spread naturally to western European saltmarshes. It is now introduced in various continents, leading to various attempts to control or eradicate the species (e.g. Hacker et al. 2001; Cottet et al. 2007). Introduced S. alterniflora is progressing along the western Atlantic coasts of France and Spain (Baumel et al. 2003; Campos et al. 2004). Another introduced Spartina species in Europe is the native South-American heptaploid species S. densiflora (Fortune et al. 2008) that is invading Mediterranean saltmarshes of the Iberian peninsula (Bortolus 2006; Castillo et al. 2008), where it hybridized with the hexaploid S. maritima (Castillo et al. 2010). In the western Mediterranean, damp depressions in dune habitats are colonized by Spartina versicolor Fabre (Fig. 1) that grows also in brackish marshes in the Atlantic Coast of the Southwest Iberian Peninsula. This species, also named Spartina juncea or Spartina durieui (Chevalier 1923; Saint-Yves 1932), had a controversial taxonomic status. It was initially recorded almost simultaneously in several Mediterranean places: first in Southern France near Agde (Fabre 1849), then in Italy (Parlatore 1848–1850), Algeria (Cosson and Maisonneuve 1867), and Portugal (Daveau 1897). In 1901, Neyraut detected this taxon on the Southwest French Atlantic coast, near Arcachon (Coste 1906). Since then, S. versicolor has established all along the western Mediteranean coasts: in Corsica (Jeanmonod and Burdet 1989) as well as on the Atlantic and Mediterranean coasts of the Iberian Peninsula (Sanche ´z-Gullo ´n2001). S. versicolor was considered as either a native Mediterranean plant (e.g. Sanche ´z-Gullo ´n2001; Giuliano and Stanisci 2010; Tison et al. 2014a,b), or an invasive species introduced from America (Sanz Elorza et al. 2004; Tison and de Foucault 2014). Based on morphological similarities, Mobberley (1956) considered S. versicolor as synonymous to S. patens, assuming that the Mediterranean populations were introduced from the Atlantic North American coast where S. patens is abundant in high marsh and dunes. Recent studies on salt marshes along the Spanish Atlantic coast have underlined the presence of S. patens in Europe (San Leon 1999; Page et al. 2010) and renewed interest in the status of S. versicolor. Prieto et al. (2011) examined three S. versicolor individuals from northern Spain (Asturias) using Internal transcribed Spacer (ITS) sequences of nuclear ribosomal DNA genes, and compared these sequences to those initially published in genus Spartina by Baumel et al. (2002) and Ferris et al. (unpublished). These individuals exhibited similar ITS sequence to S. patens, which led these authors to suggest that S. versicolor should be considered as S. patens. But it cannot be excluded that a native Mediterranean Spartina species exists besides the introduction of S. patens in Spain. Cases of cryptic invasion have already been documented in the recent history of Spartina (e.g. Bortolus et al. 2015) and can be difficult to detect (Valtuena et al. 2011). Fig. 1 Spartina versicolor avigorous population from Vieux Salins (Hye `res, France) bmitotic chromosomes counterstained with DAPI (2n =40), bar represents 5 lm Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction? 2125 123 In this study, we aim at answering the following questions: Is S. versicolor Fabre from Europe conspecific with S. patens from North America and if so is European S. patens another case of trans-Atlantic introduction into Europe? As highlighted above, worldwide invasions of Spartina are common phenomena and the Mediterranean coast may be one of the various places where American Spartina have settled after dispersal by ships during the 18th or 19th centuries. Gaining insight into the native or introduced status of S. versicolor, to its relationship with other Spartina species will be of critical importance in order to better understand the biogeography and diversification of Spartina species, as well as to determine the conservation priority level and management policy of S. versicolor in the Old World. To answer these questions, populations of S. versicolor sampled from various Mediterranean and Atlantic sites in Europe and North-Africa (including reference types from herbaria) are analyzed using cytogenetic and molecular (microsatellite, nuclear and chloroplast DNA sequences) data, and compared to North-American Spartina species. Materials and methods Plant material Fifty-seven individuals of S. versicolor were sampled from numerous Mediterranean sites (n =47) and from herbarium collections (n =10) (Table S1). The analyzed samples include specimens from various populations in France (including Corsica), Italy, Portugal, Spain and Algeria as well as the first S. versicolor plants from Agde (France) discovered by Fabre (1849). Eight Spartina patens samples were obtained from the Atlantic North American coast (Table S1). Representatives from eight other Spartina species (S. argentinensis,S. alterniflora,S. arundinacea,S. bakeri,S. densiflora,S. foliosa,S. maritima and S. pectinata) and from Sporobolus cryptandrus were additionally introduced in phylogenetic analyses. Chromosome counts The chromosome number of S. versicolor was determined on mitotic chromosomes obtained from two plants collected in France (Vieux Salins and SaintLouis du Rhone). Mitotic chromosomes were observed on metaphasic cells isolated from root tips. The roots tips of 0.5–1.5 cm length were treated with 0.04 % 8-hydroxiquinoline for 2 h at 4 °C in the dark followed by 2 h at room temperature to accumulate metaphases, then fixed in ethanol-acetic acid (3:1, v/v) for 12 h at 4 °C and stored in ethanol 70 % at -20 °C. After washing in 0.01 M enzyme buffer (citric acidsodium citrate pH 4.5) for 15 min, the roots were digested in a solution of 5 % Onozuka R-10 cellulase (Sigma) and 1 % Y23 pectolyase (Sigma) at 37 °C for 30 min. The root tips were then washed with distilled water for 30 min. Root tips transferred on a slide were squashed in a drop of 3:1 ethanol-acetic acid fixation solution. After air-drying, slides were stained with 4,6diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analysed using MetaVue TM (Universal Imaging Corporation, Downington, PA). DNA isolation, PCR amplification and DNA sequencing Genomic DNA was isolated from 100 mg of fresh (or 30 mg of herbarium) leaves from each individual using the NucleoSpin Ò Plant II Kit (Macherey– Nagel), following instructions provided by the manufacturer. DNA concentrations were estimated using the Nanodrop Spectrophotometer ND 1000 (Thermo Fischer Scientific). Four chloroplast and ten nuclear regions were amplified. Chloroplast sequences were chosen among the most variable intergenic regions identified in Poaceae (Rousseau-Gueutin et al. 2015) or within Spartina (Blum et al. 2007; Kim et al. 2013): it included the ndhC-trnV, petA-psbJ (primers designed from Rousseau-Gueutin et al. 2015), and the trnL-trnF and trnT-trnL intergenic regions (Taberlet et al. 1991). Nuclear regions included Internal transcribed Spacers (ITS) of nuclear ribosomal genes (rDNA) (White et al. 1990) and nine microsatellite markers (SSR 6, 40, 44, 72, 109, 122, 161, 172, 188) identified from S. maritima Bacterial Artificial Chromosome end sequences (Ferreira de Carvalho et al. 2013). The primer sequences used in this study are indicated in Table S2. 2126 A. Baumel et al. 123 Amplifications of chloroplast and ITS regions were carried out using the high fidelity KOD polymerase (Toyobo, Novagen) in a total volume of 50 ll. The reaction mix included 1X of KOD buffer, 1.5 mM MgSO4, 0.2 mM dNTP, 0.3 lM of each primer, 0.02 U of KOD polymerase, and 20 ng template DNA. Cycling conditions were 94 °C for 2 min, followed by 32 rounds of 94 °C for 20 s, 59.5 °C for 10 s and an extension at 70 °C for 15 s. Chloroplast and nuclear (ITS) PCR products were purified using the PCR Clean-up Gel extraction kit (Macherey–Nagel) and the purified products were sent to Macrogen Europe (Amsterdam, Netherlands) for direct sequencing. Long PCR products (from the trnT-trnL, trnL-trnF and ndhC-trnV regions) were sequenced from both sides. Sequences were cleaned and verified visually on the chromatograms (no double peaks observed). Simple Sequence Repeat (SSR) detection was performed for 60 samples (52 Old Word S. versicolor and 8 New World S. patens) using 9 microsatellite loci. Amplifications of all microsatellites were performed in 20 lL that contained 10 ng DNA, 4 lLof 5X buffer, 4 mM MgCl2, 0.2 mM dNTPs, 0.4 lMof each primer, and 0.4 units of Taq polymerase (QBiogen) in a PTC-200 Gradient Thermal Cycler (MJ Research), following touchdown PCR protocols (Migliore et al. 2013). The 50ends of the forward primers were labelled with PET or NED. The fluorescently labelled PCR products were diluted (1/40) and were separated by capillary electrophoresis, with a 500 bp size standard (LIZ500), using an ABI Prism Ò 3730xl (Applied Biosystems) automatic sequencer. Alleles were sized using PEAK SCANNER 1.0 software (Applied Biosystems). Genotyping (PCR and electrophoresis) was repeated for 8 samples to verify the reproducibility of the peak patterns. Phylogenetic analyses The data matrices generated for individual or concatenated chloroplast regions as well as the nuclear ITS matrix were obtained after aligning all sequences using Geneious (Drummond et al. 2010) and adjusting them manually. These matrices were first subjected to phylogenetic analyses using maximum parsimony. Sequence data were analyzed using PAUP* v4.0b10 (Swofford 2001) with heuristic search and the default search options. The phylogenetic analyses were performed using sequences from Sporobolus cryptandrus, a closely related species to Spartina (Peterson et al. 2014) as outgroup. Bootstrap analyses were performed with 1000 replicates (Felsenstein 1985). In addition, these data matrices were subjected to Maximum Likelihood phylogenetic analyses. The best-fitted model of sequence evolution for each region (individual or concatenated) was determined by using JModeltest (Posada 2008) implemented in MEGA 5.0 (Tamura et al. 2011). Maximum likelihood analyses were then performed for each matrix using PhyML (Guindon and Gascuel 2003), with 1000 replicates of bootstrap. Microsatellite analyses Out of the nine SSR loci, six were considered to be reliable and were subsequently analyzed. After comparisons of replicates, all dubious peaks were removed. Since most genotypes have at least three alleles in this polyploid species (see below), SSR markers were analyzed as binary data and the matrix of allele size was converted into presence/absence data matrix. Genalex 6.51 software (Peakall and Smouse 2006) was used to search for matching genotypes, bearing evidence for identical clones. The search for matching genotype was repeated accounting for one, two or three allelic errors. Allelic accumulation curves were performed (specaccum function, vegan R package, Oksanen et al. 2013) to match allelic richness of S. versicolor and S. patens. Structure of genetic diversity was analyzed using multivariate analyses of ade4 and adegenet packages of R (Dray and Dufour 2007; Jombart 2008). Principal coordinate analysis (PcoA, dudi.pco function, ade4 R package) was based on Jaccard distances (Jaccard 1901) computed on SSR presence/absence (dist.binary function, ade4 R package). For matching samples having identical genotypes (clones), only one genotype was kept in analyses based on individual genotypes and these were identified as ‘‘clones’’ on the PcoA plot. A discriminant analysis was conducted on the correlation of allele presence/absence to distinguish genetic groups according to the clustering procedure designed by Jombart et al. (2010) (DAPC analysis, adegenet R package). Finally, allele frequencies within these groups were used to compute Nei distances (Nei 1972) and to build a Neighbor Joining network to explore relationships between DAPC Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction? 2127 123 genetic groups (nj function, ape R package, Paradis et al. 2004). Results Chromosome counts revealed that in both analyzed populations, S. versicolor individuals have 2n =40 chromosomes (Fig. 1b), indicating that this taxon is a tetraploid species. No sequence heterogeneity was observed in either chloroplast or nuclear (ITS) sequences. Intra-genomic polymorphism might be expected in polyploid nuclear genomes, but the ITS regions (belonging to the rDNA gene family) seem to have undergone concerted evolution as previously found in other Spartina species (Baumel et al. 2002; Boutte et al. 2015). Maximum Parsimony (MP) analyses were performed using the ndhC-trnV (601 bp), petA-psbJ (470 bp), trnL-trnF (577 bp), trnT-trnL (631 bp), chloroplast concatenated (2278 bp) or nuclear ribosomal ITS (457 bp) matrices. These analyses resulted in 23, 34, 20, 23, 3 and 19 equal most parsimonious trees. In these analyses, S. versicolor, S. patens and S. bakeri always belonged to the same clade (Fig. 2). For the most resolved tree corresponding to the ITS regions, these three species belong to a well-supported clade (99 %) and are as positioned as a sister clade to S. arundincacea and S. densiflora (100 % bootstrap support). The sequences obtained from all S. versicolor (including the sequences obtained by Prieto et al. 2011) and S. patens samples were identical, apart from a single substitution observed in one accession of S. patens (Cheesequake state Park, Florida) for the ndhCtrnV region. S. versicolor and S. patens are closely related to S. bakeri, presenting only two substitutions (one for the ndhC-trnV and one for the ITS regions). Since similar tree topologies were obtained using Maximum Likelihood, only the MP trees are presented here. Thirty-seven alleles were recorded over the six SSR loci. All SSR genotypes were heterozygous with mostly 3 or 4 alleles per locus. Within S. versicolor, search for matching genotypes revealed 35 genotypes among 52 samples, 31 being unique and 4 being repeated from 4 to 11 times. These identical genotypes are referred as ‘‘clones’’. Accounting for one, two or three allele errors we found 41, 44 and 50 matching genotypes in S. versicolor, whereas no matching genotypes were found among the 8 samples of S. patens even accounting for 3 allele errors. Accumulation curve (Fig. 3) accounting for the unequal sampling between S. versicolor (n =52) and S. patens (n =8) revealed higher allelic diversity in S. patens than in S. versicolor: i.e. for 6 samples 32 alleles were encountered in S. patens against 26 in S. versicolor (Fig. 3). The PcoA analysis based on Jaccard distances computed between individual genotypes (Fig. 4) revealed that the main structure is due to differentiation between 6 out of 8 S. patens genotypes. The other 2 S. patens samples are more similar to S. versicolor genotypes. Herbarium specimens are scattered among S. versicolor genotypes, except one (collected in Carnon, France, in 1880 by Jouve) that is grouped with S. patens (H6, Fig. 4). The herbarium specimen collected by Fabre in (1849)inAgdehasoneofthe genotypes recorded on many individuals (‘‘matching genotypes’’ see Materials and methods) and identified as clone ‘‘b’’ (Fig. 4). This clone is found in France, Basque area, Italy and Sicily. According to the DAPC analysis (Fig. 4) and NJ network (Fig. 5), the SSR genotypes were optimally clustered in six groups. The genetic cluster number 4 (represented in green, Figs. 5, 6) was composed of European samples from France (natural populations and most herbarium samples), Italy, Corsica, and North Spain. The individuals from the southern Iberic Peninsula (south of Spain and Portugal) and Algeria are grouped in two (1 and 6) closely related clusters. Three genetic clusters could be distinguished in the American samples of S. patens:(1) Mexico-Florida-Delaware (2) New Jersey and (3) NewJersey Hampshire. The herbarium sample from France sampled in Carnon (Herault) was assigned in a group with S. patens (Mexico-Florida-Delaware group). c Fig. 2 Molecular phylogeny of Spartina based on chloroplast (andhC-trnV: 601 bp; bpetA-psbJ: 470 bp, ctrnL-trnF: 577 bp, dtrnT-trnL: 631 bp, econcatenated sequences: 2278 bp) or fnuclear ribosomal ITS sequences (457 bp) using the maximum parsimony method. For each phylogeny, one of the equally parsimonious trees that is topologically identical to the 50 % majority-rule consensus tree (petA-psbJ: 34 equally parsimonious trees; concatenated chloroplast sequences: three equally parsimonious trees; ITS: 19 equally parsimonious trees) or the 50 % majority-rule consensus tree (ndhC-trnV: 23 parsimonious trees; trnL-trnF: 20 parsimonious trees) is presented. The bootstrap percentages (1000 replicates) are shown in bold above the branches and the number of changes is indicated below. The tree is rooted using either Sporobolus cryptandrus or Sorghum bicolor 2128 A. Baumel et al. 123 5 S. bakeri S. patens (New Jersey, USA) S. versicolor (Barayo, Asturias, Spain) S. pectinata S. arundinacea S. densiflora S. alterniflora S. maritima Sporobolus cryptandrus S. foliosa S. patens (Tamaulipas, Mexico) S. patens (New Hampshire, USA) S. patens (Cheesequake state, New Jersey, USA) S. patens (Florida Desoto Park, Florida, USA) S. patens (Delaware, USA) S. patens (Bayview avenue, New Jersey, USA) S. versicolor (Pedrena, Cantabre, Spain) S. versicolor (Otur, Asturias, Spain) S. versicolor (Pantano, Sicily, Italy) S. versicolor (Boudigou de Toreilles, Pyrénées orientales, France) S. versicolor (Kouali, Tipaza, Algeria) S. versicolor (Les Aresquiers, Hérault, France) S. versicolor (Port Saint Louis, Bouches du Rhône, France) S. versicolor (Etang des Pesquiers, Var, France) 99 70 16 16 6 3 1 21 1 1 7 9 8 2 100 6 93 92 5 F S. versicolor (Prieto et al. 2011; JN133292) S. versicolor (Prieto et al. 2011; JN133290) S. versicolor (Prieto et 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. bakeri S. patens (New Jersey, USA) S. pectinata S. arundinacea S. densiflora S. alterniflora S. maritima Sorghum bicolor S. patens (Cheesequake state, New Jersey, USA) S. patens (Florida Desoto Park, Florida USA) S. patens (Delaware, USA) S. versicolor (Pantano, Sicily, Italy) S. versicolor (Boudigou de Toreilles, Pyrénées orientales, France) S. versicolor (Kouali, Tipaza, Algeria) S. versicolor (Les Aresquiers, Hérault, France) S. versciolor (Port Saint Louis, Bouches du Rhône, France) S. versciolor (Vieux salins, Var, France) S. versicolor (Guadiana river, Andalusia, Spain) S. versciolor (Banda bianca, Corsica, France) S. versicolor (Tagus estuary, Portugal) S. versicolor (Grande motte, Hérault, France) S. versciolor (Etang des Pesquiers, Var, France) S. patens (Chissahowitzka, Florida, USA) A S. bakeri S. patens (New Jersey, USA) S. pectinata S. arundinacea S. densiflora S. alterniflora S. maritima Sporobolus cryptandrus S. patens (Cheesequake state, New Jersey, USA) S. patens (Florida Desoto Park, Florida, USA) S. patens (Delaware, USA) S. versciolor (Bourdigou de Toreilles, Pyrénées orientales, France) S. versicolor (Kouali, Tipaza, Algeria) S. versicolor (Les Aresquiers, Hérault, France) S. versicolor (Port Saint Louis, Bouches du Rhône, France) S. versicolor (Vieux salins, Var, France) S. versicolor (Guadiana river, Andalusia, Spain) S. versicolor (Banda bianca, Corsica, France) S. versicolor (Tagus estuary, Portugal) S. versicolor (Grande motte, Hérault, France) S. versicolor (Etang des Pesquiers, Var, France) S. patens (Chissahowitzka, Florida, USA) S. patens (Tamaulipas, Mexico) S. patens (New Hampshire, USA) S. patens (Bayview avenue, New Jersey, USA) S. versicolor (Pedrena, Cantabre, Spain) S. versciolor (Otur, Asturias, Spain) S. versicolor (Pantano, Sicily, Italy) S. versicolor (Barayo, Asturias, Spain) S. versicolor (Ebro river, Andalusia, Spain) S. versicolor (Asperillo dunes, Andalusia, Spain) S. versicolor (Roche, Andalusia, Spain) S. versicolor (Pantano, Sicily, Italy) S. foliosa 1 64 1 1 1 1 1 1 5 3 65 96 B S. bakeri S. patens (Tamaulipas, Mexico) S. pectinata S. alterniflora Sporobolus cryptandrus S. patens (New Jersey, USA) S. patens (New Hampshire,USA) S. patens (Cheesequake state, New Jersey, USA) S. patens (Florida Desoto Park, Florida, USA) S. versicolor (Asperillo dunes, Andalusia, Spain) S. versicolor (Roche, Andalusia, Spain) S. versicolor (Pantano, Sicily, Italy) S. versicolor (Bourdigou de Toreilles, Pyrénées orientales, France) S. versicolor (Kouali, Tipaza, Algeria) S. versicolor (Banda bianca, Corsica, France) S. foliosa S. maritima S. densiflora S. gracilis S. arundinacea 67 3 1 1 2 7 4 3 2 2 3 88 C S. bakeri S. pectinata S. arundinacea S. densiflora S. alterniflora S. maritima Sporobolus cryptandrus S. patens (Cheesequake state, New Jersey, USA) S. versciolor (Bourdigou de Toreilles, Pyrénées orientales, France) S. versicolor (Kouali, Tipaza, Algeria) S. versicolor (Les Aresquiers, Hérault, France) S. versicolor (Port Saint Louis, Bouches du Rhône, France) S. versicolor (Vieux salins, Var, France) S. versicolor (Guadiana river, Andalusia, Spain) S. versicolor (Tagus estuary, Portugal) S. versicolor (Grande motte, Hérault, France) S. versicolor (Etang des Pesquiers, Var, France) S. versicolor (Pedrena, Cantabre, Spain) S. versciolor (Otur, Asturias, Spain) S. versicolor (Pantano, Sicily, Italy) S. versicolor (Ebro river, Andalusia, Spain) S. versicolor (Asperillo dunes, Andalusia, Spain) S. versicolor (Roche, Andalusia, Spain) S. versicolor (Pantano, Sicily, Italy) S. foliosa 1 1 1 2 2 1 1 2 4 9 53 91 94 D S. bakeri S. patens (Cheesequake state, New-Jersey USA) S. versicolor (Bourdigou de Toreilles, Pyrénées orientales, France) S. pectinata S. arundinacea S. densiflora S. alterniflora S. maritima Sorghum bicolor 100 85 67 66 54 68 85 12 5 6 9 3 3 3 3 2 1 1 1 0 E Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction? 2129 123 The S. patens cluster being the most similar to S. versicolor (NJ network Fig. 6) is observed all along the Atlantic North American coast from Mexico to Delaware. The cluster 4 which has most of the S.versicolor samples, and the herbarium sample collected by Fabre, are the most similar to S. patens according to the NJ network (Fig. 6). Discussion Our results reveal that S. versicolor is a tetraploid species with 40 chromosomes, as found in S. patens (Marchant 1968) and no European or North-African populations analyzed can be differentiated genetically from North American S. patens samples as they exhibit similar rDNA ITS and cpDNA sequences. In his monograph of Spartina, Mobberley (1956) stressed the morphological similarities between these two taxa, although several phenotypes were described for S. patens (Mobberley 1956). Our results support the hypothesis that all European and African populations of S. versicolor are in fact North American S. patens introduced before or at the beginning of the nineteenth-century. Although some microsatellite variation was detected between S. patens and S. versicolor, only few genotype differences were observed. Genetic differences regarding microsatellite alleles would most likely result from intraspecific genetic diversity in S. patens-versicolor populations; this is supported by genetic similarities between some North-American S. patens and S. versicolor samples as can be seen on PcoA results (Fig. 4). The introduction origin could be in the areas covered by the genetic cluster 4 (Fig. 5), i.e. France, North Spain or Italy because the corresponding genotype is the most similar to S. patens in the NJ network (Fig. 6). This pattern is also clear in the PcoA analysis (Fig. 4). The S. versicolor samples from Portugal and South Spain, or Algeria are either derived from this introduction or resulted from a second introduction. Spartina patens is a highly variable rhizomatous species, exhibiting high ecological amplitude along the Atlantic coast of North America from Canada to Central America, colonizing high salt marsh zones, beaches and sand dunes, with variable seed set (Silander and Antonovics 1979). Allozyme studies in native S. patens populations (Silander 1984) revealed important polymorphism with a decreased role of vegetative reproduction from dune to marsh habitats. In contrast microsatellite genotyping revealed reduced genetic diversity in S. versicolor compared to North American S. patens samples (Fig. 3), which is consistent with a genetic bottleneck following introduction in Europe together with the predominant clonal propagation of the introduced plants. Indeed we found 17 matching SSR genotypes within S. versicolor but this number increased to 41 when accounting for one allele error indicating that genetic variation within S. versicolor could be mainly of somaclonal variation. Preliminary surveys in S. versicolor populations revealed sterile pollen (R. Amirouche, unpublished data), which is in agreement with the observation that this taxon rarely produces seeds (Fabre 1849; San Leon et al. 1999; Tison et al. 2014; our personal observations). Further sampling and phylogeographic analyses are needed in the native region of S. patens to better document the history of this taxon and to identify the precise populations that were introduced in the Mediterranean. Although various studies have documented distribution, ecology and plasticity of S. patens in North America (e.g. Frasco and Good 1982; Burdick and Mendelssohn 1987; Burdick et al. 1989; Foote and Reynolds 1997; Lonard et al. 2010), very few studies have documented genetic diversity in S. patens (e.g. Wu 2012) and there is a great need to develop DNA-based analyses at the genome level in the native range of this species, which plays an important ecological role, preventing coastal erosion and being used in dune restoration. Samples Alleles 123456 20 25 30 35 40 S. patens Fig. 3 Rarefaction curves of allelic diversity for the seven SSR loci 2130 A. Baumel et al. 123