Molecular Phylogenetics and Evolution 61 (2011) 659–670

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Molecular Phylogenetics and Evolution

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Molecular phylogeny and systematics of the highly polymorphic Rumexbucephalophorus complex (Polygonaceae)

M. Talavera a,⇑, F. Balao a, R. Casimiro-Soriguer a, M.Á. Ortiz a, A. Terrab a, M. Arista a, P.L. Ortiz a,T.F. Stuessy b, S. Talavera a

a Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apdo. 1095, 41080 Sevilla, Spainb Institute of Botany, University of Vienna, Rennweg 14, A-1030 Wien, Austria

a r t i c l e i n f o

Article history:Received 14 October 2010Revised 9 June 2011Accepted 4 August 2011Available online 16 August 2011

Keywords:AFLPITSBayesian population analysisDiaspore traitsMediterranean BasinMacaronesian area

1055-7903/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ympev.2011.08.005

⇑ Corresponding author.E-mail address: mtalavera@us.es (M. Talavera).

a b s t r a c t

Rumex bucephalophorus is a very polymorphic species that has been subjected to various taxonomic stud-ies in which diverse infraspecific taxa have been recognised on the basis of diaspore traits. In this studywe used molecular markers (ITS and AFLP) to explore this remarkable diversity, to test previous hypoth-eses of classification, and attempt to explain biogeographic patterns. Results show that R. bucephalopho-rus forms a monophyletic group in which diversification began around 4.2 Mya, at the end of MessinianSalinity Crisis. The two molecular markers clearly show a deep divergence separating subsp. bucephalo-phorus from all other subspecific taxa, among which subsp. canariensis also constitutes a separate andwell distinguishable unit. In contrast, subspecies hispanicus and subsp. gallicus constitute a monophyleticgroup in which three subgroups can be recognised: subsp. hispanicus, subsp. gallicus var. gallicus andsubsp. gallicus var. subaegeus. However, these three subgroups are not clearly distinguished geneticallyor morphologically, so that in formal classification it would be preferable to treat them at the varietallevel.

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1. Introduction

The genus Rumex comprises around 200 species mostly distrib-uted in Europe and North America. The genus has had a compli-cated taxonomic history. Löve and Kapoor (1967) split Rumexinto four separate genera: Rumex L. and Bucephalophora Pau, bothwith annual or perennial, generally hermaphrodite species, andAcetosa Miller and Acetosella Fourr., both with predominantly suff-ruticose, and dioecious, gynodioecious, polygamous or rarely her-maphrodite species. In their treatment, the genus Rumex s.s.includes c. 150 species, mostly polyploids, with the base numberx = 10; Bucephalophora is a monospecific genus with the speciesBucephalophora aculeata (L.) Pau (a synonym of Rumex bucephalo-phorus L.), which is diploid, x = 8; Acetosa includes c. 35 species,is diploid or polyploid, with x = 9, 10; and Acetosella with five spe-cies, is diploid or polyploid, x = 7, 8, rarely 4 or 5. These four generarecognised by Löve and Kapoor (1967) were previously treated asinfrageneric taxa of Rumex by the majority of authors; as sectionsby Meissner (1856) and Willkomm (1862), and as subgenera byRechinger (1964) and López González (1990), with the name sect.Platypodium Willk. [or subg. Platypodium (Willk.) Rech. fil.] substi-tuting for Bucephalophora.

ll rights reserved.

The molecular phylogenetic study of Rumex s.l. by Navajas-Pérez et al. (2005) did not provide support for the four-genus viewproposed by Löve and Kapoor (1967). Rather, they defined twolarge clades within the genus Rumex, one formed by the speciesof subg. Rumex, and the other composed of the species of subgen-era Acetosa, Acetosella and Platypodium. Within this latter clade,which diversified around 15–16 Mya, the dioecious species of sub-genera Acetosa and Acetosella formed a monophyletic group thatwas sister to subg. Platypodium, with its sole species, R. bucephalo-phorus (Navajas-Pérez et al., 2005).

R. bucephalophorus is widely distributed throughout the coast-lands of the Mediterranean Basin and the Atlantic coast of SW Eur-ope (W Iberian Peninsula and SW France), NW Morocco andMacaronesia (Azores, Madeira and Canary Archipelago). Althoughcoastal or sub-coastal populations are the most frequent, inlandpopulations also occur throughout the distribution of the species.R. bucephalophorus is an annual, except for the high mountain pop-ulations on the Island of Madeira, which are perennial and suffrut-escent (var. fruticescens); the populations are hermaphrodite orgynomonoecious, self-incompatible, and pollination is anemophi-lous (Talavera, 2011). The species is remarkably heterocarpic andup to four functionally different diaspore types are produced: bur-ied diaspores with thick pedicels at the plant base (BD; Fig. 1A) andderived from female flowers, and three diaspore types from her-maphrodite flowers situated along the aerial stems (Talavera

Fig. 1. Morphology of the four diaspore types known in the Rumex bucephalophorus complex. (A) Buried diaspore; (B) fixed diaspore; (C) short-dispersible diaspore; (D) long-dispersible diaspore.

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et al., 2010). The latter comprise non-dispersible, fixed diasporeswith thick pedicels (FD; Fig. 1B), and two types of dispersiblediaspores, one type with short and thin pedicels (SD; Fig. 1C),and the other type with long thin pedicels (LD: Fig. 1D). The lattertwo types of diaspores are dispersed mainly by wind or by epizo-ochory, although hydrochory is also a possibility (Talavera et al.,unpub. data).

Due to the high morphological variability found in R. bucephalo-phorus, up to seven subspecies have been recognised, and the com-plex has been subjected to various taxonomic studies (Steinheil,1838; Rechinger, 1939; Press, 1988). The most recent treatment(Press, 1988) recognises four subspecies: subsp. bucephalophorus(found throughout Mediterranean coastlands except in Morocco),subsp. hispanicus (Steinh.) Rech. fil. (NW Iberian Peninsula andSW France), subsp. canariensis (Steinh.) Rech. fil. (Madeira and Can-ary Islands) and subsp. gallicus (Steinh.) Rech. fil. (throughout Med-iterranean basin, W Iberian Peninsula, W Morocco andMacaronesia). Remarkably, subsp. gallicus is sympatric with theremaining subspecies and all of them are inter-compatible (Talave-ra, unpub. results) and have the same chromosome number(2n = 16; Löve and Kapoor, 1967; García et al., 1989). Press(1988) distinguished the subspecies by the morphology of boththe pedicel and the valves of the diaspores: subsp. bucephalophorushas both long-dispersible and fixed diaspores, always bigger thanthose of the other subspecies and with 2–3 pairs of wide teeth ineach valve; subsp. hispanicus has short-dispersible diaspores with4–6 pairs of uncinate teeth in each valve; subsp. canariensis hasshort-dispersible diaspores with valves that are entire or with 4–8 pairs of straight teeth; and subsp. gallicus has both fixed diasp-ores with entire valves and long dispersible diaspores with toothedvalves. In some populations of subsp. gallicus, plants can also havefruits at the base of the stems and such populations have been re-ferred to var. subaegeus Maire in the W Mediterranean. However,these differences among subspecies are often less evident in thefield and recognition of these taxa can be difficult due to the vari-ability found in natural populations.

In this study we used molecular markers to evaluate the infra-specific differentiation in R. bucephalophorus and to infer a phylog-eographic scenario. As a framework for our initial studies, and toassist our collection of material, we used the infraspecific classifi-cation by Press (1988). Our study addresses the following ques-tions: (1) Is R. bucephalophorus a monophyletic complex? (2) Ifso, is it possible to distinguish genetically different infraspecifictaxa within this species? (3) Can we propose a phylogeny-basedclassification in R. bucephalophorus? and (4) Can we elucidate acoherent phylogeography of R. bucephalophorus?

We used two approaches to answer these questions: thesequencing of nuclear ribosomal regions (internal transcribedspacers, nrITS), and a more variable DNA fingerprint method(amplified fragment length polymorphism, AFLP). ITS has beenused successfully in the study of several old Tertiary lineages, such

as Anthyllis montana (Kropf et al., 2002), the Campanula lusitanicacomplex (Cano-Maqueda et al., 2008), and Erophaca baetica(Casimiro-Soriguer et al., 2010). To test the monophyly of the R.bucephalophorus complex we selected species from the differentclades reported by Navajas-Pérez et al. (2005) in their phylogenyof the genus Rumex. If the clade including R. bucephalophorus isas old as estimated by Navajas-Pérez et al. (2005), ITS sequencescould be a useful tool to distinguish genetically well-defined taxa.In contrast, AFLP markers are highly polymorphic and have beenused successfully in differentiating among infraspecific taxa (Ter-rab et al., 2008) or closely related species (Tremetsberger et al.,2004; Ortiz et al., 2009; Balao et al., 2010). Moreover, AFLP markersare among the most commonly used in phylogeographic studies inrecent years (Beheregaray, 2008).

2. Material and methods

2.1. Plant Material and DNA isolation

We focused our sampling in the western area of distribution ofR. bucephalophorus since this is where the four subspecies recogni-sed by Press (1988) occur. A total of 204 individuals were sampledfrom 20 populations from N Africa (Atlantic and MediterraneanMorocco and Algeria), the Iberian Peninsula (Atlantic and SWcoastlands and some inland localities), and Macaronesia (Table 1,Fig. 2). Employing the morphological criteria for subspeciesadopted by Press (1988), these samples included 25 plants from3 populations of subsp. bucephalophorus, 39 from 4 populationsof subsp. canariensis (one of which, pop 7, belonged to var. frutices-cens), 53 from 5 populations of subsp. hispanicus, and 87 from 8populations of subsp. gallicus (41 individuals from 4 populationsof var. gallicus and 46 plants from 4 populations of var. subaegeus).In each population diaspore traits were recorded for ten plants.

Fresh leaves were collected at random from 5 to 12 individualsper population, and dried in silica gel until processed for DNA iso-lation. Vouchers are deposited in the Herbarium of the Universityof Seville, Spain (SEV). Total genomic DNA, for nrDNA ITS and AFLPanalyses, was extracted following the CTAB protocol (Doyle andDoyle, 1987) with modifications (Terrab et al., 2007). All sampledpopulation extracts were checked for the presence and amountof DNA on 1% TAE agarose gels.

2.2. nrDNA ITS

We randomly chose two individuals per population to analysethe nuclear ribosomal Internal Transcribed Spacer regions ITS1and ITS2; to do that we used universal primers ITS4 and ITS5(White et al., 1990). ITS1 and ITS2 were amplified using the samemethodology as in Casimiro-Soriguer et al. (2010). We encoun-tered a number of problems in sequencing ITS from Rumex. Amongthe 40 initial samples, 20 presented multiple rDNA arrays probably

Table 1Population-based estimates of genetic diversity and diaspore types in Rumex bucephalophorus. Total no. of fragments (Fragtot); % polymorphic fragments (Fragpoly); average genediversity (HD); private fragments (Fragpriv), values given in brackets if they are fixed; rare fragments index (DW). Averages for subspecies are shown in bold. Asterisks indicatesequenced samples for ITS variation. Diaspore type: BD = buried diaspore, FD = fixed diaspore, SD = short-dispersible diaspore, LD = long-dispersible diaspore. Vouchers for each ofthese populations are deposited at SEV. The numbered locations in the table are also plotted in map on the Fig. 2.

Taxa/localities Diaspore type Coordinates Collector no N Fragtot Fragpoly HD Fragpriv DW FST

a subsp. bucephalophorus 0.301. Algeria. Skikda. Kef Fatima SD, LD 36�540/7�020 EV: 242/07 5 100 90.0 0.10 1 7.52. Spain. Málaga. Marbella SD, LD 36�290/�4�460 MT: 363/06 11 120 82.5 0.08 2 28.03. Spain. Cádiz. La Línea⁄ FD, SD 36�140/�5�190 MT: 10/07 9 119 87.3 0.09 5 24.0

Mean 8.3 113 86.6 0.09 2.7 19.8

b subsp. canariensis (Steinh.) Rech. fil. 0.53b1 var. canariensis

4. Madeira. Porto Santo island FD, SD 33�050/�16�180 MS:195/07 11 144 81.2 0.10 7(1) 28.25. Madeira I. Seixal⁄ FD, SD 32�480/�17�030 MS:196/07 7 99 66.7 0.06 3 12.86. Madeira II. Garajau⁄ FD, SD 32�380/�16�510 MS: 194/07 11 126 76.2 0.08 4 23.1

b2 var. fruticescens (Bornm.) J.R. Press7. Madeira III. Pico Arieiro⁄ FD, SD 32�440/�16�560 MS:285/07 10 118 78.8 0.08 3 18.8

Mean 9.7 121.7 75.7 0.08 4.2 20.7

c subsp. hispanicus (Steinh.) Rech. fil. 0.218. Spain. Lugo. San Cosme⁄ FD, SD 43�330/�7�110 ST: 306/06 12 190 93.7 0.15 6 27.19. Spain. Pontevedra I. El Tojar FD, SD 42�170/�8�500 SC: 287/07 7 178 89.9 0.16 1 11.210. Spain. Pontevedra II. Moaña⁄ FD, SD 42�160/�8�450 SC: 288/07 12 187 93.0 0.14 1 18.511. Portugal. Minho. Viana do Castello FD, LD 41�430/�8�520 ST: 267/06 10 180 93.9 0.15 1 17.112. Portugal. Estremadura. Peniche BD, FD, LD 39�200/�9�210 ER: 224/07 12 181 94.5 0.14 0 14.1

Mean 10.6 183.2 93.0 0.15 1.8 17.6

d subsp. gallicus (Steinh.) Rech. fil. 0.31d1. var. gallicus 0.22

13. Spain. Segovia. Villar de Sobrepeña FD, LD 41�160/�3�480 MM: 225/07 8 115 96.5 0.10 0 4.314. Spain. Salamanca. Pte de la Salud FD, LD 40�570/�5�400 ER: 223/07 9 95 93.7 0.08 0 4.415. Spain. Granada. Hoya de Guadix FD,LD 37�360/�3�080 JP: 221/07 12 181 95.0 0.14 1 18.816. Morocco. Alhucemas FD, LD 35�110/�3�580 ST: 61/07 12 163 98.2 0.13 3 17.9

Mean 10.2 138.5 95.8 0.11 1 11.3

d2. var. subaegeus Maire 0.2617. Spain. Huelva. Acebrón BD, FD, LD 37�070/�6�310 MT: 4/07 12 126 94.4 0.10 0 9.318. Spain. Sevilla. Paradas⁄ BD, FD, LD 37�160/�5�270 MT: 13/07 11 122 89.3 0.09 1 7.819. Morocco. Larache BD, FD, LD 35�080/�6�080 MT: 158/07 12 182 90.1 0.14 2 20.820. Morocco. Kenitra⁄ BD, FD, LD 34�130/�6�340 MT: 145/07 11 174 87.4 0.12 0 16.1

Mean 11.5 151 90.3 0.11 0.7 13.5

Mean 10.9 144.7 93.1 0.11 0.9 12.4

EV: E. Vela; MT: M. Talavera; MS: M. Sequeira; ST: S. Talavera; SC: S. Castroviejo; ER: E. Rico; MM: M. Martínez; JP: J. Peña.

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as consequence of incomplete concerted evolution. In another 12,only 200 base pairs (bp) were amplified, likely due to diversecauses, such as presence of pseudogenes or secondary structure(Álvarez and Wendel, 2003; Nieto Feliner and Roselló, 2007). Asa consequence, we obtained complete ITS1 and ITS2 sequencesfor only 8 of the 40 individuals. Fortunately, these successful sam-ples represented each of the four subspecies recognised by Press(1988), viz. three individuals of subsp. canariensis, two of subsp.hispanicus, two of subsp. gallicus, and one of subsp. bucephalopho-rus (Genbank Nos. HQ389207–HQ389222). The aligned data matrixare deposited in the TreeBASE database (Accession No. S11620).

We constructed a phylogeny using our ITS sequences along withGenBank accessions of related species belonging to the other threesubgenera of Rumex. We selected species from the different cladesof Rumex reported by Navajas-Pérez et al. (2005). Specifically, weincluded one species from subgenus Acetosella, the dioecious Ru-mex graminifolius (accession number: AJ831539, AJ844277); threespecies from subgenus Acetosa, the dioecious Rumex acetosa(AJ580774, AJ580790), the hermaphrodite–polygamous Rumexinduratus (AJ580778, AJ580794) and the polygamous–gynodioe-cious Rumex hastatus (AF338218); and three hermaphrodite spe-cies from subgenus Rumex, Rumex conglomeratus (AJ580785,

AJ580789); Rumex japonicus (AF338220) and Rumex aquitanicus(AJ810986, AJ810996). We included Fallopia convolvulus(AF040064) as outgroup based on the phylogeny by Navajas-Pérezet al. (2005). To construct the phylogeny we used maximum parsi-mony analysis (PAUP� ver. 4.0 beta 10), and full heuristic searchoption (10,000 replicates) to calculate bootstrapping values (Swof-ford, 2002).

We estimated divergence times from the phylogeny, eitherwithin R. bucephalophorus or from its ancestors, using the Bayesianrelaxed molecular clock method implemented in BEAST 1.4.7(Drummond and Rambaut, 2007). Because Rumex belongs to aclade with a poor fossil record, the clock was calibrated by a ‘‘uni-form prior’’ on two nodes previously estimated by Navajas-Pérezet al. (2005): 16–15 Mya for the appearance of dioecy in Rumex(Fig. 3, node A), and 13–12 Mya as the divergence time betweenthe dioecious species of the subgenera Acetosella (R. graminifolius)and Acetosa (R. acetosa) (Fig. 3, node B). Furthermore, the hermaph-rodite, polygamous or gynodioecious species of subgenera Acetosa(R. hastatus and R. induratus) and Rumex (R. conglomeratus, R. japo-nicus and R. aquitanicus) were included within the ingroup, while F.convolvulus was used to root the tree. We also assumed uncorrela-tion, Yule model for speciation, and automatic tuning of operators.

Fig. 2. Geographical distribution of the studied populations of Rumex bucephalophorus. Populations are labelled according to their number and taxonomy (see Table 1).Triangle: subsp. bucephalophorus; open hexagon: subsp. canariensis var. canariensis, filled hexagon: subsp. canariensis var. fruticescens; square: subsp. hispanicus; open circle:subsp. gallicus var. gallicus, filled circle: subsp. gallicus var. subaegeus.

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Analyses were run for 100 million generations (sampling every1000th), with a burn-in of one million generations.

2.3. AFLPs

All 204 sampled individuals were included in this study. Fol-lowing the protocols established by Vos et al. (1995), genomicDNA (0.5 lg on average) was digested with two restriction endo-nucleases (EcoRI and MseI), the fragments ligated to double-stranded adaptors (EcoRI and MseI) at 37 �C for 2 h, then diluted20-fold with TE0.1 buffer. Fragments with matching nucleotideswere amplified (downstream of the restriction sites) using prese-lective primers based on EcoRI and MseI adaptors. This resultedin a �16-fold decrease in the number of fragments amplified. Pre-selective and selective amplifications were performed in a thermalcycler (Gene Amp� PCR system 9700, PE Applied Biosystems). Inorder to assess the reliability of the method, a random fraction(N = 22, i.e., 10.8%) of the samples was analysed twice from the firstPCR. It was found that duplicate analyses were largely indistin-guishable, with a 98% repeatability of bands.

A screening of selective primers (consisting of a battery of 72)was run on eight individuals from eight populations, and the threeprimers that generated the most polymorphic fragments were se-lected. These were: MseI-CAC/EcoRI-AGC, MseI-CAT/EcoRI-ACG,MseI-CAG/EcoRI-ACT. Fluorescence-labelled selective amplifica-tion products were separated on a 3130xl Genetic Analyzers (Ap-plied Biosystems) capillary sequencer with an internal sizestandard (GeneScan-500 ROX, PE Applied Biosystems). Alignmentof the raw data with the size standard was performed with ABIPrism GeneScan software 2.1 (PE Applied Biosystems). AFLP frag-ments were scored for bands 80–500 bp in length with softwaredeveloped at Montana State University (Genographer ver. 1.6.0,

available at http://hordeum.oscs.montana.edu/genographer) usingthe ‘thumbnail’ option. Results were summarised as a matrix ofpresence/absence.

2.3.1. AFLP-based genetic diversity and population differentiationTotal number of fragments (Fragtot), percentage of polymorphic

fragments (Fragpoly) as well as the private fragment number (Frag-priv), were determined for each population, using FAMD version1.08 (Schlüter and Harris, 2006). Mean genetic diversity (HD) wascomputed with Arlequin version 3.01 (Excoffier et al., 2005). TheRarity Index (equivalent to the frequency of down-weighted mar-ker values; i.e., DW) was also calculated with AFLPdat (Ehrich,2006) and R Statistical Software ver. 2.8.1 (R Development CoreTeam).

The extent of genetic differentiation, measured as FST (the fixa-tion index) as well as shared exclusive fragments (Fragpriv) wasdetermined both for pairs of populations and taxa using Arlequin3.01 (Excoffier et al., 2005). The relationship between pairwiseFST values and geographical distances was investigated with Man-tel tests based on Spearman rank correlations with 1 million per-mutations (package ade4 in R Statistical Software; Dray andDufour, 2007).

2.3.2. Plant clusteringAmong-plant genetic distances (Nei and Li, 1979) were calcu-

lated from the matrix of AFLP scores and represented graphicallywith an unrooted neighbour-joining dendrogram built withSPLITSTREE 4.6 (Huson and Bryant, 2006). The bootstrap supportfor each branch was calculated with PAUP� ver. 4.0 beta 10 (Swof-ford, 2002; neighbour-joining clustering, 10,000 replicates).

We applied a principal co-ordinate analysis (PCoA) of a Euclid-ean matrix of similarities among individuals to investigate geneticdistances and relationships between individuals. The PCoA was

Fig. 3. Bayesian chronogram based on a maximum parsimony tree obtained from nuclear rDNA sequences (ITS1, ITS2) of Rumex bucephalophorus, and close relatives; Fallopiaconvolvulus was chosen as the outgroup. The nodes used to calibrate the clock are marked with black dots ((A) 16–15 Mya appearance of dioecy in genus Rumex, and (B) 13–12 divergence between R. acetosa–R.graminifolius, see Navajas-Pérez, 2005). � = populations of R. bucephalophorus subsp. hispanicus (8 and 10).

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performed with the program FAMD version 1.08 (Schlüter andHarris, 2006) and plotted with SPSS 15.0. Genetic relationshipsamong populations were further explored with a Bayesian assign-ment technique implemented in STRUCTURE 2.2.3 (Pritchard et al.,2000; Falush et al., 2003) and the analysis was carried out at thefreely available Bioportal (http://www.bioportal.uio.no/). Thisapproach uses individual multilocus genotypes to constructclusters of genetically similar plants, in a way that maximisesHardy Weinberg equilibrium and minimises linkage disequilib-rium within clusters. Following Falush et al. (2003) and Evannoet al. (2005) we used the ‘admixture’ and the ‘correlated allelefrequency’ models, a combination judged appropriate when theaim is to infer subtle population structures, as expected in ourdataset. To ensure convergence of the Monte Carlo Markov Chain(MCMC) we used 50,000 burn-ins followed by 500,000 iterations.The true number of genetically distinct sets of populations wasdetermined from analyses that had varying values of K, from 1 toa maximum of 20 (i.e., the total number of populations). Tenindependent runs were performed and averaged for each value ofK. Furthermore, the partitioning (within and among populationsand taxa) of genetic variation was determined with an Analysisof Molecular Variance (AMOVA; Excoffier et al., 2005) performedover all the studied samples. The effect on genetic variation ofsubspecies was also studied with AMOVA.

3. Results

3.1. Diaspore distribution

In R. bucephalophorus subsp. bucephalophorus fixed non-dispers-ible diaspores (FD), long-dispersible diaspores (LD) and short-dis-persible diaspores (SD) were found, but buried diaspores (BD)were always absent (Table 1). In subsp. canariensis both fixed andshort-dispersible diaspores were recorded, in subsp. gallicus bur-ied, fixed and long-dispersible diaspores were found, and lastly,in subsp. hispanicus the four diaspore types were found (Table 1).In subsp. bucephalophorus each diaspore internal bract always pre-sents two pairs of teeth. In the other three subspecies internalbracts can be entire or toothed; when toothed, in subsp. canariensisthey had 3–5 pairs, in subsp. hispanicus 3–4 and in subsp. gallicus 3.

3.2. nrDNA ITS phylogeny

The complete nrDNA ITS region in R. bucephalophorus rangedfrom 384 to 387 base pairs (bp). Maximum parsimony analysisgenerated 2 equally parsimonious trees that were similar, but inone the R. bucephalophorus complex is most closely related to theR. hastatus–R. induratus clade (without bootstrap support), and in

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the other (shown in Fig. 3) it appears most closely related to the R.acetosa–R. graminifolius clade (also without bootstrap support). Thelength of the tree shown in Fig. 3 was 375 steps, the consistencyindex (CI) excluding autopomorphies was 0.80, and the retentionindex (RI) was 0.73. Our results show that R. bucephalophorus(subg. Platypodium), subg. Acetosa (R. acetosa, R. hastatus and R.induratus) and subg. Acetosella (R. graminifolius) form a well sup-ported monophyletic clade (98% Bootstrap, BS).

All samples of R. bucephalophorus form a robust monophyleticgroup (99% BS) in which subsp. bucephalophorus is sister to allother subspecies, but with a very low support (51% BS, 11 nucleo-tide changes), whilst subsp. canariensis forms a strongly supportedclade (81% BS), differing from the subsp. hispanicus plus subsp. gal-licus clade in 5 nucleotide changes; var. fruticescens (pop.7, Fig. 3)differs from var. canariensis in 2 changes.

According to the relaxed molecular clock, R. bucephalophorusbegan to diversify around 4.2 (95% HPD: 8.4–1.1) Mya into twophylogenetic lineages, one comprising R. bucephalophorus subsp.bucephalophorus, and the other the ancestral taxon of the threeother subspecies (Fig. 3). Subspecies canariensis diverged at0.7 Mya (95% HPD: 1.8–0.1) and var. fruticescens (a perennial) at0.3 Mya (95% HPD: 0.9–0.01).

3.3. AFLP-based genetic diversity and population differentiation

In 204 individuals of R. bucephalophorus, a total of 437 unambig-uously scoreable DNA fragments were detected, of which 428(97.9%) were polymorphic. The number of fragments for each pri-mer combination was MseI-CAC/EcoRI-AGC: 132, MseI-CAT/EcoRI-ACG: 141, and MseI-CAG/EcoRI-ACT: 164. This number of frag-ments was sufficient to distinguish all 204 individuals as separatephenotypes.

The populations of subsp. hispanicus presented the highestmean values of genetic diversity (HD = 0.15), those of subsp. gallicusshowed intermediate values (HD = 0.11) and the populations ofsubsp. bucephalophorus and subsp. canariensis presented the lowestvalues (HD = 0.09 and 0.08, respectively; Table 1). The highest per-centage of polymorphic fragments was found in subsp. hispanicusand gallicus (93%) and the lowest in subsp. canariensis (75.7%; Ta-ble 1). The highest numbers of private fragments, exclusive to each

Table 2Matrix of pairwise fixation index (AMOVA-derived FST) and pairwise shared private fragmeFST values are in bold, and empty cells denote that the number of shared fragments equal

Subsp. bucephalophorus Subsp. canariensis Subsp. hispanicus

1 2 3 4 5 6 7a 8 9 101 –2 0.37 – 93 0.26 0.28 – 14 0.60 0.69 0.65 – 2 3 25 0.66 0.73 0.70 0.53 – 16 0.62 0.71 0.68 0.45 0.58 – 37 0.63 0.71 0.67 0.54 0.58 0.52 –8 0.48 0.60 0.56 0.58 0.57 0.61 0.59 – 1 29 0.46 0.59 0.55 0.57 0.56 0.60 0.59 0.22 –10 0.47 0.59 0.55 0.57 0.55 0.60 0.59 0.28 0.09 –11 0.45 0.60 0.55 0.55 0.54 0.58 0.58 0.29 0.16 0.1912 0.44 0.58 0.54 0.54 0.51 0.56 0.55 0.28 0.15 0.1513 0.37 0.57 0.50 0.56 0.56 0.59 0.56 0.35 0.33 0.3314 0.45 0.61 0.56 0.61 0.61 0.63 0.62 0.39 0.37 0.3815 0.36 0.53 0.47 0.49 0.49 0.52 0.51 0.34 0.31 0.3216 0.35 0.53 0.48 0.53 0.50 0.55 0.51 0.37 0.33 0.3517 0.48 0.61 0.57 0.61 0.61 0.62 0.62 0.40 0.35 0.3618 0.50 0.63 0.59 0.61 0.61 0.64 0.62 0.41 0.34 0.3619 0.45 0.58 0.55 0.57 0.56 0.59 0.57 0.38 0.31 0.3320 0.49 0.61 0.58 0.60 0.60 0.62 0.60 0.41 0.35 0.38

a Var. fruticescens.

population (Fragpriv), and the highest values of rare fragment index(DW) were found in populations of subsp. canariensis and buceph-alophorus (Table 1). Only one population (pop. 4 of subsp. canarien-sis) showed one fixed private fragment, i.e., this fragment wasfound in all the individuals of the population. The highest fixationindex value was found in subsp. canariensis (FST = 0.53) and thelowest in subsp. hispanicus (FST = 0.21; Table 1).

The genetic dissimilarity between any two populations (as FST)significantly increased with increasing spatial distance (Manteltest, r = 0.518, N = 20, p = 0.002). The pairwise FST distances andthe exclusive shared fragments (Table 2) agreed with the resultsmentioned above, showing that most populations diverged mark-edly from each other. The exceptions were found mainly in subsp.hispanicus in which four populations (among the five studied)showed low genetic distances (FST) to each other (pop. 9, 10, 11and 12; Table 2).

High values of FST were found between subsp. bucephalophorusand subsp. canariensis and also the other subspecies (0.48–0.35;Table 3). In contrast, the lowest FST value (0.15) was found betweensubsp. hispanicus and subsp. gallicus (Table 3). Interestingly, thetwo latter subspecies shared a markedly high number of pairwiseprivate fragments (Fragpriv = 79, Table 3), and subsp. gallicus shareda considerable number of pairwise private fragments with theother subspecies (20 and 24, Table 3). In contrast, subsp. hispanicusand subsp. canariensis only shared three private fragments. Thehighest number of private fragments was found in subsp. canarien-sis (39, Table 3). When considering the two varieties of subsp. gal-licus, the FST value between both varieties (0.20) is very similar tothe values between subsp. hispanicus and these varieties (0.18 withvar. gallicus and 0.19 with var. subaegeus; data not shown). In addi-tion, the number of pairwise private fragments shared by bothvarieties (11) is less than those between subsp. hispanicus andthese varieties (18 with var. gallicus and 13 with var. subaegeus;data not shown).

3.4. AFLP-based plant clustering

The neighbour-joining unrooted dendrogram of AFLPs resolvedsubsp. bucephalophorus as a separate cluster with 100% BS (Fig. 4).In the other large cluster, composed of the other three subspecies,

nts (Fragpriv, above diagonal) for the 20 Rumex bucephalophorus populations. Extremes zero.

Subsp. gallicus var. gallicus Subsp. gallicus var. subaegeus

11 12 13 14 15 16 17 18 19 201 1

12 1

11

2 11

1 2–0.21 – 1 10.33 0.26 –0.35 0.33 0.19 –0.33 0.24 0.14 0.28 – 1 10.35 0.27 0.19 0.31 0.18 – 1 2 10.36 0.33 0.32 0.34 0.35 0.38 –0.36 0.30 0.31 0.33 0.33 0.36 0.22 –0.33 0.30 0.34 0.36 0.33 0.35 0.30 0.28 – 60.36 0.33 0.39 0.41 0.36 0.38 0.36 0.32 0.05 –

Table 3Matrix of pairwise shared private fragments (values above diagonal), and pairwise fixation index (amova-derived FST, euclidean distance) (values below diagonal), and privatefragments (Fragpriv) of each subspecies of Rumex bucephalophorus based on the analysis of 437 AFLP markers.

Subsp. bucephalophorus Subsp. canariensis Subsp. hispanicus Subsp. gallicus

Subsp. bucephalophorus – 5 5 20Subsp. canariensis 0.48 – 3 24Subsp. hispanicus 0.42 0.37 – 79Subsp. gallicus 0.35 0.32 0.15 –Fragpriv 25 39 25 28

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subsp. canariensis (99% BS) and subsp. hispanicus (76% BS) werealso strongly supported, but subsp. gallicus was not a cohesivegroup (Fig. 4).

The Principal Coordinate analysis (PCoA), based on Euclideansimilarity matrix, showed that subsp. bucephalophorus and subsp.canariensis constituted separate and well distinguishable units(Fig. 5). The other two subspecies were clustered much closer to-gether, but there was a trend to distinguish the plants into threegroups: subsp. hispanicus, subsp. gallicus var. subaegeus and subsp.gallicus var. gallicus (Fig. 5). In fact, a Bayesian analysis conductedwith STRUCTURE showed that K = 3 or K = 5 were the most likelynumber of clusters in our data set (Fig. 6). In both cases the subsp.canariensis and subsp. bucephalophorus formed separate groups.With K = 3, populations of the subsp. hispanicus and subsp. gallicusjoined together, while with K = 5, populations of the subsp. hispani-cus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeusappeared as distinct groups. Furthermore, this Bayesian analysisassigned the majority of individuals to their populations or groupsof populations with a high probability.

The results of analyses of molecular variance (AMOVA) showedthat when no groups were considered, differences among plants ac-counted for 52.7% of variation (Table 4a). When the populationswere grouped, the model considering three groups (subsp. bucepha-lophorus, subsp. canariensis, and subsp. gallicus + hispanicus), ex-plained the largest fraction of variation (30.87%, Table 4b),followed by that considering five groups (subsp. bucephalophorus,subsp. canariensis, subsp. hispanicus, subsp. gallicus var. gallicusand subsp. gallicus var. subaegeus) which explained 28.66%(Table 4d) of variation. The model considering four groups (the foursubspecies) explained a lower percentage of variation (Table 4c).

4. Discussion

4.1. Diaspore distribution

The peduncle and the position of the diaspore of R. bucephalo-phorus determine its ultimate function. Diaspores with thickpeduncles (buried and fixed) remain attached to the plant untilsenescence and thus, they do not disperse. In contrast, diasporeswith very thin peduncles (long- and short-dispersible) are easilydetached from the plant and are dispersed by wind or epizoochory(Talavera, 2011). These four diaspore types can also differ in themorphology of the bracts (differing in number of teeth and in bractlength) (López González, 1990). Thus, we can differentiate diasp-ores according to their function and/or morphology. All these dia-spore traits have been traditionally used to distinguishinfraspecific taxa in R. bucephalophorus (Press, 1988; López Gon-zález, 1990). However, we have found that subsp. bucephalophoruscan produce up to three functional diaspore types (fixed non-dis-persible, long-dispersible and short-dispersible), subsp. gallicuscan produce buried, fixed and long-dispersible diaspores, and theremaining two subspecies can produce all four diaspore types.Although plants of subsp. canariensis studied here neither pro-

duced buried nor long-dispersible diaspores, a wider sampling inanother study showed that these diaspores can be found in somepopulations of this taxon (Talavera, 2011). However, neither popu-lations nor plants of each subspecies simultaneously present allthese types of diaspores. On the basis of diaspore morphology,subsp. bucephalophorus is the only one to be clearly distinguished:they are the biggest and have two pairs of wide triangular teeth. Inthe remaining taxa there is an overlapping number of teeth on thebracts and so this trait cannot be used to distinguish these taxa.Thus, our results clearly show that separation of all the infraspe-cific taxa of R. bucephalophorus cannot rely on these traits.

4.2. nrDNA ITS phylogeny

Although ITS sequences were obtained for only eight of the 20populations of R. bucephalophorus studied, the phylogenetic struc-ture was consistent with data from AFLP markers for 204 individ-uals across all 20 populations.

ITS analyses clearly show the monophyly of the clade compris-ing the Acetosa–Acetosella–Platypodium subgenera (Fig. 3, 98% BS).Given that the majority of species in this clade are currently dis-tributed around the Mediterranean Basin, we can assume that R.bucephalophorus originated in this area at 15–16 Mya during theMiocene (Navajas-Pérez et al., 2005) although its infraspecificdiversification began much later (Fig. 3). According to our relaxedmolecular clock analysis, R. bucephalophorus began to diversifyaround 4.2 Mya into two phylogenetic lineages comprising R.bucephalophorus subsp. bucephalophorus, and the ancestor of theother three subspecies. From the group formed by the other taxa,only subsp. canariensis is robustly supported and estimated to havediverged during the Pleistocene, at about 0.7 Mya. Thus, resultsfrom ITS clearly show that subsp. bucephalophorus and subsp.canariensis are well differentiated taxa, whereas subsp. gallicusand subsp. hispanicus constitute a single group (see Fig. 3).

The R. bucephalophorus lineage is estimated to have originatedabout 15 Mya, with extant taxa diversifing around 4 Mya (Fig. 3).During this time, the western margin of the embryonic Mediterra-nean had a very complicated geological history (Rosenbaum et al.,2002) with shifting islands, a reduced SE Iberian land area, and theBetic and Rifian sea corridors that gave access to the Atlantic. Thelatter closed between 7.16 and 6 Mya (Krijgsman et al., 1999a;Seidenkrantz et al., 2000), thus causing the gradual desiccation ofthe Mediterranean Basin (7.2–5.3 Mya) named the MessinianSalinity Crisis (Hsü et al., 1973; Bocquet et al., 1978; Krijgsmanet al., 1999b; Duggen et al., 2003), and leading to marked climaticchanges (Rouchy and Caruso, 2006; Murphy et al., 2009). Our dataindicate that towards the end of this complicated period, the firstdiversification of R. bucephalophorus took place, with subsp.bucephalophorus as sister to a clade with the ancestor of theremaining subspecies. Thus, our results are in accordance withother phylogenetic studies of animals (García-París and Jockusch,1999; Sanmartín, 2003; Fromhage et al., 2004; Martínez-Solanoet al., 2004) and plants (Caujapé-Castells and Jansen, 2003; Hampe

Fig. 4. NJ dendrogram of the 204 individuals of Rumex bucephalophorus analysed for AFLPs, based on Nei and Li’s genetic distance. Bootstrap values (10,000 replicates), ifhigher than 50%, are indicated at each node. For details of populations, see Table 1.

666 M. Talavera et al. / Molecular Phylogenetics and Evolution 61 (2011) 659–670

et al., 2003; Terrab et al., 2007, 2008; Cano-Maqueda et al., 2008)with a Mediterranean distribution. This provides evidence that

the end of the Messinian played an important role in their expan-sion and diversification.

Fig. 5. Ordination of AFLP data of Rumex bucephalophorus. Principal CoordinateAnalysis (PCoA) based on a matrix of Euclidean similarity among the 204 studiedindividuals.

M. Talavera et al. / Molecular Phylogenetics and Evolution 61 (2011) 659–670 667

4.3. AFLP-based genetic diversity, population differentiation, and plantclustering

The early divergence between subsp. bucephalophorus and theother subspecies is also supported by AFLP analyses that show thatthe genetic distance between this subspecies and the other subspe-cies is always very high (FST = 0.35–0.48; see Table 3). In fact, subsp.bucephalophorus is the only subspecies that can be clearly distin-

Fig. 6. Genetic structure of Rumex bucephalophorus inferred by Bayesian clustering of AFLindividual is represented by a vertical bar coloured according to the assigned group(s). Ththe most stable and likely assignments estimated by structure (at K = 3 and K = 5) (see

guished morphologically as the diaspores are consistently largeand with wide triangular teeth (López González, 1990). Moreover,the two populations of subsp. bucephalophorus from the Iberian Pen-insula indeed have high DW values (pops. 2 and 3, Table 1), stronglysuggesting they are old (Stehlik, 2002; Schönswetter and Tribsch,2005). In contrast, the population of subsp. bucephalophorus fromAlgeria (pop. 1, Table 1) is among the most genetically impoverishedof the species, suggesting that this is a recently established popula-tion, but it could also reflect diverse stochastic processes, such as ge-netic drift or bottlenecks (Ellstrand and Elam, 1993).

The subspecies gallicus and hispanicus recognised by Press(1988) were not strongly supported by the molecular markers em-ployed here. The maximum parsimony tree generated by the ITSdata and the NJ dendrogram generated by the AFLP data indicatethat the group of populations analysed from R. bucephalophorus fallinto three groups, two of which coincide with subspecies bucepha-lophorus and canariensis, but the third group includes populationsassigned to subsp. gallicus and subsp. hispanicus. However, the dis-tinct clustering analyses, especially PCoA and STRUCTURE (Figs. 5and 6; respectively), show that within this third group, three sub-groups of populations can be distinguished. These subgroups cor-respond to the populations that were assigned to subsp.hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var.subaegeus, which differ in geographical distribution and diasporetraits (Press, 1988). It is notable that populations of subsp. hispani-cus, subsp. gallicus var. subaegeus and subsp. gallicus var. gallicusshowed similar genetic distances among them (Table 2). This sug-gests that the three subgroups should be considered at the sametaxonomic level, but since they are not as clearly separated assubsp. bucephalophorus and subsp. canariensis, we think that it isbetter to consider them as three varieties of a single subspecies,for which the name subsp. gallicus has nomenclatural priority.

Most populations of hispanicus–gallicus group have high geneticdiversity and high indices of rare fragments (Table 1); these factspoint towards an ancient origin of these populations (Bonin

P data. Assignment of 204 individuals into K genetically distinguishable groups. Eache 20 populations are identified following Table 1. The upper and lower bands show

text).

Table 4Results of analyses of molecular variance (AMOVA) of AFLP data from 20 Rumex bucephalophorus populations. The studied populations (1–20, see Table 1) were grouped in threedifferent ways (b–d). All models were highly significant (P < 0.00001).

Grouping of populations and source of variation Df Variance component Variation (%) FST (95%CI)

a Global analysis 0.47 (0.45–0.49)Among populations 19 22.15 47.30Among plants 184 24.68 52.70

b [1–3][4–7][8–20]Among groups 2 17.07 30.87 0.31 (0.28–0.34)Among populations 17 13.55 24.50Among plants 184 24.68 44.63

c [1–3][4–7][8–12][13–20]Among groups 3 14.02 27.76 0.28 (0.24–0.30)Among populations 16 11.81 23.38Among plants 184 24.68 48.86

d [1–3][4–7][8–12][13–16][17–20]Among groups 4 14.09 28.66 0.29 (0.26–0.31)Among populations 15 10.41 21.16Among plants 184 24.68 50.18

668 M. Talavera et al. / Molecular Phylogenetics and Evolution 61 (2011) 659–670

et al., 2007). The exceptions are pops. 13 and 14 from the SpanishCentral Plateau of subsp. gallicus var. gallicus, and pops. 17 and 18from the Guadalquivir valley (SW Iberian Peninsula) of subsp. gal-licus var. subaegeus. Given that coastal or sub-coastal zones are themain habitats for the hispanicus–gallicus group, it is likely that theinland populations are more recently founded. The Central Plateaupopulations appear to be derived from Betic and Rifean popula-tions (pops. 15 and 16, Fig. 2) because, as shown by our results (Ta-ble 2), they are genetically very close, with markedly low geneticdistances (FST) between both groups of populations (pops. 13 and14 vs. 15 and 16; Table 2). The low genetic diversity (HD, Fragpoly,and DW; Table 1) of Central Plateau populations of subsp. gallicusvar. gallicus (pops. 13 and 14) suggests a very recent origin derivedfrom southern populations (pops. 15 and 16). The south to northmigration of the plants of Spain has been recently documented inother species such as Ferula loscosii (Pérez-Collazos et al., 2009).The Guadalquivir valley populations of subsp. gallicus var. subaeg-eus (pops. 17 and 18, Fig. 2) probably originated from the popula-tions of subsp gallicus var. subaegeus of NW Morocco (pops. 19 and20, Fig. 2) since they are genetically similar (Fig. 4) and NW Mor-occo populations have higher genetic diversity than Guadalquivirvalley populations (Table 1). In fact, there are numerous examplesof species that crossed from the Atlantic coast of N Africa to theIberian peninsula during the Pliocene–Pleistocene (Pardo et al.,2008; Ortiz et al., 2009; Casimiro-Soriguer et al., 2010).

The remaining taxon, subspecies canariensis, was very well sup-ported by AFLP data as it has a very high number of private frag-ments (39, Table 3) and its genetic distance to the other taxa isalso high (FST = 0.32–0.48, Table 3). According to our results(Fig. 3), subsp. canariensis diverged recently (0.73 Mya), presum-ably as a consequence of the arrival on Macaronesia of immigrantsfrom continental populations during the early Pleistocene. The factthat subsp. canariensis and subsp. gallicus share the highest numberof pairwise private fragments (Table 3), and have the lowest FST va-lue (Table 3), indicate that subsp. canariensis is most closely relatedto subsp. gallicus. On the other hand, populations of subsp. canari-ensis are very diverse and genetic distances among them are veryhigh. Reproductive isolation of populations is common in speciesthat occur on oceanic volcanic islands where the immigrantsunderwent a strong adaptive radiation (Sanmartín et al., 2008).This could explain the strong genetic differences between the pop-ulation of Porto Santo Island and the three populations of MadeiraIsland (Table 2). However, the three populations of Madeira Islandalso showed markedly high genetic distances (Table 2). Multiple

colonisation events on Madeira Island could explain these differ-ences. Moreover, the orography of the island is very rugged andthese populations although geographically close could also bereproductively isolated. Two populations (5 and 6) are on oppositesides of the island separated by a high mountain range and thethird population (pop. 7) is in the high part of this range. The latterconsists of perennial woody plants and is the only locality where R.bucephalophorus exhibits this habit. This supports the idea thatthese populations of Madeira are isolated, because gene exchangebetween them would have eliminated or spread the perennial ha-bit. These perennial woody plants were previously treated assubsp. fruticescens (Rechinger, 1939), and subsequently recognisedas a variety of subsp. canariensis (Press, 1988). Both annual andsuffruticose species occur in the genus Rumex, but it is unusualfor both habits to appear in the same species. There is ample sug-gestion based on morphology of the genus Rumex to support theview that the annual habit is usually derived from perennial(Löve and Kapoor, 1967). However, according to our molecularclock analyses, var. fruticescens diverged only some 0.32 Mya(Fig. 3), thus suggesting that in R. bucephalophorus the suffruticosehabit evolved from the annual condition. This pattern has been alsoreported in numerous molecular phylogenetic plant studies in oce-anic islands, such as in the Hawaian archipelago (Baldwin et al.,1991), and in Macaronesia, especially in the Canary archipelago(Sang et al., 1995; Böhle et al., 1996; Kim et al., 1996; Mes andHart, 1996; Panero et al., 1999; Thiv et al., 1999; Helfgott et al.,2000; Barber et al., 2002; Mansion et al., 2009). In fact, the rever-sion from annual to perennial seems to be genetically simple, asdemonstrated in Arabidopsis thaliana (Melzer et al., 2008).

In conclusion, despite the fact that R. bucephalophorus originatedin the Miocene, its subspecific diversification is likely to have coin-cided with the end of Messinian Salinity Crisis; this supports the evi-dence that this geological period played an important role in thediversification of many plant species. The two molecular markerswe employed indicate that R. bucephalophorus is a monophyleticspecies, and also clearly differentiate subsp. bucephalophorus as sis-ter to all other subspecies, and subsp. canariensis as the most re-cently derived subspecific taxon; subsp. hispanicus and subsp.gallicus constitute a monophyletic group, within which three sub-groups can be recognised that correspond to subsp. hispanicus,subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus. Giventhat, at present, these subgroups are not clearly separated, neithergenetically nor morphologically, we propose that it would be betterto consider all three as varieties of subsp. gallicus.

M. Talavera et al. / Molecular Phylogenetics and Evolution 61 (2011) 659–670 669

Acknowledgments

This work was supported with FEDER funds and grants from theSpanish Ministerio de Ciencia y Tecnología to P.L.O. (CGL2008-02533-E), to S.T. (CGL2009-08178) and to M.A. (CGL2005-0195;CGL2008-02531-E; CGL2009-08257) and a PhD Grant from theSpanish Ministerio de Educación y Ciencia to M.T (BES-2006-13925). The authors thank collectors indicated in Table 1, Dr. P.E.Gibbs for English revision and comments on the Ms. M.J. Arizaand L. Navarro for technical support, and to the staff of the BiologyLaboratory Service (CITIUS, Seville University).

References

Álvarez, I., Wendel, J.F., 2003. Ribosomal ITS sequences and plant phylogeneticinference. Molecular Phylogenetics and Evolution 29, 417–434.

Balao, F., Valente, L.M., Vargas, P., Herrera, J., Talavera, S., 2010. Radiative evolutionof polyploid races of the Iberian carnation Dianthus broteri (Caryophyllaceae).New Phytologist 187, 542–551.

Baldwin, B., Kyhos, D., Dvorak, J., Carr, G., 1991. Chloroplast DNA evidence for aNorth American origin of the Hawaiian silversword alliance (Asteraceae).Proceedings of the National Academy of Sciences of the United States ofAmerica 88, 1840–1843.

Barber, J.C., Francisco-Ortega, J., Santos-Guerra, A., Turner, K.G., Jansen, R.K., 2002.Origin of Macaronesian Sideritis L. (Lamioideae: Lamiaceae) inferred fromnuclear and chloroplast sequence datasets. Molecular Phylogenetics andEvolution 23, 293–306.

Beheregaray, L.B., 2008. Twenty years of phylogeography: the state of the fieldand the challenges for the Southern Hemisphere. Molecular Ecology 17,3754–3774.

Bocquet, G., Widler, B., Kiefer, H., 1978. The Messinian Model. A new outlook for thefloristics and systematics of the Mediterranean area. Candollea 33, 269–287.

Böhle, U., Hilger, H., Martin, W., 1996. Island colonization and evolution of theinsular woody habit in Echium L. (Boraginaceae). Proceedings of the NationalAcademy of Sciences of the United States of America 93, 11740–11745.

Bonin, A., Ehrich, D., Manel, S., 2007. Statistical analysis of amplified fragmentlength polymorphism data: a toolbox for molecular ecologists andevolutionists. Molecular Ecology 16, 3737–3758.

Cano-Maqueda, J., Talavera, S., Arista, M., Catalán, P., 2008. Speciation andbiogeographical history of the Campanula lusitanica complex (Campanulaceae)in the Western Mediterranean region. Taxon 57, 1252–1266.

Casimiro-Soriguer, R., Talavera, M., Balao, F., Terrab, A., Herrera, J., Talavera, S., 2010.Phylogeny and genetic structure of Erophaca (Leguminosae), a East–WestMediterranean disjunct genus from the Tertiary. Molecular Phylogenetics andEvolution 56, 441–450.

Caujapé-Castells, J., Jansen, R.K., 2003. The influence of the Miocene Mediterraneandesiccation on the geographical expansion and genetic variation ofAndrocymbium gramineum (Cav.) McBride (Colchicaceae). Molecular Ecology12, 1515–1525.

Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities offresh leaf tissue. Phytochemical Bulletin 19, 11–15.

Dray, S., Dufour, A., 2007. The ade4 package: implementing the duality diagram forecologists. Journal of Statistical Software 22, 1–20.

Drummond, A., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis bysampling trees. BMC Evolutionary Biology 7, 214.

Duggen, S., Hoernle, K., van den Bogaard, P., Rupke, L., Morgan, J.P., 2003. Deep rootsof the Messinian salinity crisis. Nature 422, 602–606.

Ehrich, D., 2006. AFLPdat: a collection of R functions for convenient handling ofAFLP data. Molecular Ecology Notes 6, 603–604.

Ellstrand, N.C., Elam, D.R., 1993. Population genetic consequences of smallpopulation size: implications for plant conservation. Annual Review ofEcology and Systematics 24, 217–242.

Evanno, G., Regnaut, S., Goudet, J., 2005. Detecting the number of clusters ofindividuals using the software structure: a simulation study. Molecular Ecology14, 2611–2620.

Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin ver. 3.0: an integrated softwarepackage for population genetics data analysis. Evolutionary BioinformaticsOnline 1, 47–50.

Falush, D., Stephens, M., Pritchard, J., 2003. Inference of population structure usingmultilocus genotype data: linked loci and correlated allele frequencies. Genetics164, 1567–1587.

Fromhage, L., Vences, M., Veith, M., 2004. Testing alternative vicariance scenarios inWestern Mediterranean discoglossid frogs. Molecular Phylogenetics andEvolution 31, 308–322.

García, C., Pastor, J., Luque, T., 1989. Contribución al estudio cariológico del géneroRumex (Polygonaceae). Acta Botanica Malacitana 14, 129–140.

García-París, M., Jockusch, E.L., 1999. A mitochondrial DNA perspective on theevolution of Iberian Discoglossus (Amphibia: Anura). Journal of Zoology 248,209–218.

Hampe, A., Arroyo, J., Jordano, P., Petit, R.J., 2003. Rangewide phylogeography of abird-dispersed Eurasian shrub: contrasting Mediterranean and temperateglacial refugia. Molecular Ecology 12, 3415–3426.

Helfgott, D., Francisco-Ortega, J., Santos-Guerra, A., Jansen, R., Simpson, B., 2000.Biogeography and breeding system evolution of the woody Bencomia alliance(Rosaceae) in Macaronesia based on ITS sequence data. Systematic Botany 25,82–97.

Hsü, K.J., Ryan, W.B.F., Cita, M.B., 1973. Late Miocene desiccation of theMediterranean. Nature 242, 240–244.

Huson, D., Bryant, D., 2006. Application of phylogenetic networks in evolutionarystudies. Molecular Biology and Evolution 23, 254–267.

Kim, S., Crawford, D., Francisco-Ortega, J., Santos-Guerra, A., 1996. A common originfor woody Sonchus and five related genera in the Macaronesian islands:molecular evidence for extensive radiation. Proceedings of the NationalAcademy of Sciences of the United States of America 93, 7743–7748.

Krijgsman, W., Hilgen, F.J., Raffi, I., Sierro, F.J., Wilson, D.S., 1999a. Chronology, causesand progression of the Messinian salinity crisis. Nature 400, 652–655.

Krijgsman, W., Langereis, C.G., Zachariasse, W.J., Boccaletti, M., Moratti, G., Gelati, R.,Iaccarino, S., Papani, G., Villa, G., 1999b. Late Neogene evolution of the Taza-Guercif Basin (Rifian Corridor, Morocco) and implications for the Messiniansalinity crisis. Marine Geology 153, 147–160.

Kropf, M., Kadereit, J.W., Comes, H.P., 2002. Late Quaternary distributional stasis inthe submediterranean mountain plant Anthyllis montana L. (Fabaceae) inferredfrom ITS sequences and amplified fragment length polymorphism markers.Molecular Ecology 11, 447–463.

López González, G., 1990. Género Rumex L. In: Castroviejo, S., Laínz, M., LópezGonzález, G., Montserrat, P., Muñoz Garmendia, F., Paiva, J., Villar, L. (Eds.), Floraiberica. Real Jardín Botánico, C.S.I.C., Madrid, pp. 595–634.

Löve, Á., Kapoor, B., 1967. A chromosome atlas of the collective genus Rumex.Cytologia 32, 328–342.

Mansion, G., Selvi, F., Guggisberg, A., Conti, E., 2009. Origin of Mediterranean insularendemics in the Boraginales: integrative evidence from molecular dating andancestral area reconstruction. Journal of Biogeography 36, 1282–1296.

Martínez-Solano, I., Gonçalves, H.A., Arntzen, J.W., García-París, M., 2004.Phylogenetic relationships and biogeography of midwife toads(Discoglossidae: Alytes). Journal of Biogeography 31, 603–618.

Meissner, K.F., 1856. Rumex. In: Candolle, A.P.D. (Ed.), Prodromus, vol. 14.Sumptibus Victoris Masson, Parisiis, pp. 41–74.

Melzer, S., Lens, F., Gennen, J., Vanneste, S., Rohde, A., Beeckman, T., 2008.Flowering-time genes modulate meristem determinacy and growth form inArabidopsis thaliana. Nature Genetics 40, 1489–1492.

Mes, T., Hart, H., 1996. The evolution of growth-forms in the Macaronesian genusAeonium (Crassulaceae) inferred from chloroplast DNA RFLPs and morphology.Molecular Ecology 5, 351–363.

Murphy, L., Kirk-Davidoff, D., Mahowald, N., Otto-Bliesner, B., 2009. A numericalstudy of the climate response to lowered Mediterranean Sea level during theMessinian salinity crisis. Palaeogeography, Palaeoclimatology, Palaeoecology279, 41–59.

Navajas-Pérez, R., de la Herrán, R., González, G.L., Jamilena, M., Lozano, R., Rejón,C.R., Rejón, M.R., Garrido-Ramos, M.A., 2005. The evolution of reproductivesystems and sex-determining mechanisms within Rumex (Polygonaceae)inferred from nuclear and chloroplastidial sequence data. Molecular Biologyand Evolution 22, 1929–1939.

Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in termsof restriction endonucleases. Proceedings of the National Academy of Sciencesof the United States of America 76, 5269–5273.

Nieto Feliner, G., Roselló, J.A., 2007. Better the devil you know? Guidelines forinsightful utilization of nrDNA ITS in species-level evolutionary studies inplants. Molecular Phylogenetics and Evolution 44, 911–919.

Ortiz, M.Á., Tremetsberger, K., Stuessy, T.F., Terrab, A., García-Castaño, J.L., Talavera,S., 2009. Phylogeographic patterns in Hypochaeris section Hypochaeris(Asteraceae, Lactuceae) of the western Mediterranean. Journal ofBiogeography 36, 1384–1397.

Panero, J., Francisco-Ortega, J., Jansen, R., Santos-Guerra, A., 1999. Molecularevidence for multiple origins of woodiness and a New World biogeographicconnection of the Macaronesian Island endemic Pericallis (Asteraceae:Senecioneae). Proceedings of the National Academy of Sciences of the UnitedStates of America 96, 13886–13891.

Pardo, C., Cubas, P., Tahiri, H., 2008. Genetic variation and phylogeography ofStauracanthus (Fabaceae, Genisteae) from the Iberian Peninsula and northernMorocco assessed by chloroplast microsatellite (cpSSR) markers. AmericanJournal of Botany 95, 98–109.

Pérez-Collazos, E., Sánchez-Gómez, P., Jiménez, J.F., Catalán, P., 2009. Thephylogeographical history of the Iberian steppe plant Ferula loscosii (Apiaceae):a test of the abundant-centre hypothesis. Molecular Ecology 18, 848–861.

Press, J.R., 1988. Intraspecific variation in Rumex bucephalophorus L. BotanicalJournal of the Linnean Society 97, 344–355.

Pritchard, J.K., Stephens, M., Donnelly, P., 2000. Inference of population structureusing multilocus genotype data. Genetics 155, 945–959.

Rechinger, K.H., 1939. Versuch einer natürlichen Gliederung des Formenkreises vonRumex bucephalophorus L. Botaniska Notiser 485, 504.

Rechinger, K.H., 1964. The genus Rumex L. In: Tutin, T.G., Heywood, V.H., Burges,N.A., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea, vol. 2.University Press, Cambridge, pp. 82–89.

Rosenbaum, G., Lister, G., Duboz, C., 2002. Relative motions of Africa, Iberia andEurope during Alpine orogeny. Tectonophysics 359, 117–129.

Rouchy, J., Caruso, A., 2006. The Messinian salinity crisis in the Mediterraneanbasin: a reassessment of the data and an integrated scenario. SedimentaryGeology 188, 35–67.

670 M. Talavera et al. / Molecular Phylogenetics and Evolution 61 (2011) 659–670

Sang, T., Crawford, D., Stuessy, T., Mario Silva, O., 1995. ITS sequences and thephylogeny of the genus Robinsonia (Asteraceae). Systematic Botany 20, 55–64.

Sanmartín, I., 2003. Dispersal vs. vicariance in the Mediterranean: historicalbiogeography of the Palearctic Pachydeminae (Coleoptera, Scarabaeoidea).Journal of Biogeography 30, 1883–1897.

Sanmartín, I., van der Mark, P., Ronquist, F., 2008. Inferring dispersal: a Bayesianapproach to phylogeny-based island biogeography, with special reference to theCanary Islands. Journal of Biogeography 35, 428–449.

Schlüter, P.M., Harris, S.A., 2006. Analysis of multilocus fingerprinting data setscontaining missing data. Molecular Ecology Notes 6, 569–572.

Schönswetter, P., Tribsch, A., 2005. Vicariance and dispersal in the alpine perennialBupleurum stellatum L. (Apiaceae). Taxon 54, 725–732.

Seidenkrantz, M.S., Kouwenhoven, T.J., Jorissen, F.J., Shackleton, N.J., van der Zwaan,G.J., 2000. Benthic foraminifera as indicators of changing Mediterranean-Atlantic water exchange in the late Miocene. Marine Geology 163, 387–407.

Stehlik, I., 2002. Glacial history of the alpine herb Rumex nivalis (Polygonaceae): acomparison of common phylogeographic methods with nested clade analysis.American Journal of Botany 89, 2007–2016.

Steinheil, A.D., 1838. Matériaux pour servir à la Flore de Barbarie. Annales desSciences Naturelles, Paris, Série 2 (9), 193–211.

Swofford, D.L., 2002. PAUP�: Phylogenetic Analysis using Parsimony (� and otherMethods), Version 4, Sunderland, Massachusetts.

Talavera, M., Ortiz, P.L., Arista, M., Berjano, R., Imbert, E., 2010. Disentanglingsources of maternal effects in the heterocarpic species Rumex bucephalophorus.Perspectives in Plant Ecology, Evolution and Systematics 12, 295–304.

Talavera, M., 2011. La heterocarpia en Rumex bucephalophorus L.: significadoecológico e implicaciones filogeográficas. Ph. Doctoral Thesis. University ofSeville.

Terrab, A., Schönswetter, P., Talavera, S., Vela, E., Stuessy, T.F., 2008. Range-widephylogeography of Juniperus thurifera L., a presumptive keystone species ofwestern Mediterranean vegetation during cold stages of the Pleistocene.Molecular Phylogenetics and Evolution 48, 94–102.

Terrab, A., Talavera, S., Arista, M., Paun, O., Stuessy, T.F., Tremetsberger, K., 2007.Genetic diversity at chloroplast microsatellites (cpSSRs) and geographicstructure in endangered West Mediterranean firs (Abies spp., Pinaceae). Taxon56, 409–416.

Thiv, M., Struwe, L., Kadereit, J., 1999. The phylogenetic relationships and evolutionof the Canarian laurel forest endemic Xanthus viscosus (Aiton) Griseb.(Gentianaceae): Evidence from matK and ITS sequences, and floralmorphology and anatomy. Plant Systematics and Evolution 218, 299–317.

Tremetsberger, K., Talavera, S., Stuessy, T.F., Ortiz, M.Á., Weiss-Schneeweiss, H.,Kadlec, G., 2004. Relationship of Hypochaeris salzmanniana (Asteraceae,Lactuceae), an endangered species of the Iberian Peninsula, to H. radicata andH. glabra and biogeographical implications. Botanical Journal of the LinneanSociety 146, 79–95.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van De Lee, T., Hornes, M., Frijters, A., Pot,J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNAfingerprinting. Nucleic Acids Research 23, 4407–4414.

White, T., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing offungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide toMethods and Applications 18, 315–322.

Willkomm, H.M., 1862. Polygoneae. In: Willkomm, H.M., Lange, J. (Eds.), ProdromusFlorae Hispanicae, vol. 1. Sumtibus E. Schweizerbart (E. Koch), Stuttgartiae, pp.279–292.