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

Molecular Phylogenetics and Evolution 61 (2011) 659–670

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Molecular phylogeny and systematics of the highly polymorphic Rumex bucephalophorus 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 b

Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apdo. 1095, 41080 Sevilla, Spain 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 2010 Revised 9 June 2011 Accepted 4 August 2011 Available online 16 August 2011 Keywords: AFLP ITS Bayesian population analysis Diaspore traits Mediterranean Basin Macaronesian area

a b s t r a c t Rumex bucephalophorus is a very polymorphic species that has been subjected to various taxonomic studies in which diverse infraspecific taxa have been recognised on the basis of diaspore traits. In this study we used molecular markers (ITS and AFLP) to explore this remarkable diversity, to test previous hypotheses of classification, and attempt to explain biogeographic patterns. Results show that R. bucephalophorus forms a monophyletic group in which diversification began around 4.2 Mya, at the end of Messinian Salinity Crisis. The two molecular markers clearly show a deep divergence separating subsp. bucephalophorus from all other subspecific taxa, among which subsp. canariensis also constitutes a separate and well distinguishable unit. In contrast, subspecies hispanicus and subsp. gallicus constitute a monophyletic group in which three subgroups can be recognised: subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus. However, these three subgroups are not clearly distinguished genetically or morphologically, so that in formal classification it would be preferable to treat them at the varietal level. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction The genus Rumex comprises around 200 species mostly distributed in Europe and North America. The genus has had a complicated taxonomic history. Löve and Kapoor (1967) split Rumex into four separate genera: Rumex L. and Bucephalophora Pau, both with annual or perennial, generally hermaphrodite species, and Acetosa Miller and Acetosella Fourr., both with predominantly suffruticose, and dioecious, gynodioecious, polygamous or rarely hermaphrodite species. In their treatment, the genus Rumex s.s. includes c. 150 species, mostly polyploids, with the base number x = 10; Bucephalophora is a monospecific genus with the species Bucephalophora aculeata (L.) Pau (a synonym of Rumex bucephalophorus L.), which is diploid, x = 8; Acetosa includes c. 35 species, is diploid or polyploid, with x = 9, 10; and Acetosella with five species, is diploid or polyploid, x = 7, 8, rarely 4 or 5. These four genera recognised by Löve and Kapoor (1967) were previously treated as infrageneric taxa of Rumex by the majority of authors; as sections by Meissner (1856) and Willkomm (1862), and as subgenera by Rechinger (1964) and López González (1990), with the name sect. Platypodium Willk. [or subg. Platypodium (Willk.) Rech. fil.] substituting for Bucephalophora. ⇑ Corresponding author. E-mail address: [email protected] (M. Talavera). 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.08.005

The molecular phylogenetic study of Rumex s.l. by NavajasPérez et al. (2005) did not provide support for the four-genus view proposed by Löve and Kapoor (1967). Rather, they defined two large clades within the genus Rumex, one formed by the species of subg. Rumex, and the other composed of the species of subgenera Acetosa, Acetosella and Platypodium. Within this latter clade, which diversified around 15–16 Mya, the dioecious species of subgenera Acetosa and Acetosella formed a monophyletic group that was sister to subg. Platypodium, with its sole species, R. bucephalophorus (Navajas-Pérez et al., 2005). R. bucephalophorus is widely distributed throughout the coastlands of the Mediterranean Basin and the Atlantic coast of SW Europe (W Iberian Peninsula and SW France), NW Morocco and Macaronesia (Azores, Madeira and Canary Archipelago). Although coastal or sub-coastal populations are the most frequent, inland populations also occur throughout the distribution of the species. R. bucephalophorus is an annual, except for the high mountain populations on the Island of Madeira, which are perennial and suffrutescent (var. fruticescens); the populations are hermaphrodite or gynomonoecious, self-incompatible, and pollination is anemophilous (Talavera, 2011). The species is remarkably heterocarpic and up to four functionally different diaspore types are produced: buried diaspores with thick pedicels at the plant base (BD; Fig. 1A) and derived from female flowers, and three diaspore types from hermaphrodite flowers situated along the aerial stems (Talavera

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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) longdispersible diaspore.

et al., 2010). The latter comprise non-dispersible, fixed diaspores with thick pedicels (FD; Fig. 1B), and two types of dispersible diaspores, one type with short and thin pedicels (SD; Fig. 1C), and the other type with long thin pedicels (LD: Fig. 1D). The latter two types of diaspores are dispersed mainly by wind or by epizoochory, although hydrochory is also a possibility (Talavera et al., unpub. data). Due to the high morphological variability found in R. bucephalophorus, up to seven subspecies have been recognised, and the complex 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 and SW France), subsp. canariensis (Steinh.) Rech. fil. (Madeira and Canary Islands) and subsp. gallicus (Steinh.) Rech. fil. (throughout Mediterranean basin, W Iberian Peninsula, W Morocco and Macaronesia). Remarkably, subsp. gallicus is sympatric with the remaining subspecies and all of them are inter-compatible (Talavera, 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 both the pedicel and the valves of the diaspores: subsp. bucephalophorus has both long-dispersible and fixed diaspores, always bigger than those of the other subspecies and with 2–3 pairs of wide teeth in each valve; subsp. hispanicus has short-dispersible diaspores with 4–6 pairs of uncinate teeth in each valve; subsp. canariensis has short-dispersible diaspores with valves that are entire or with 4– 8 pairs of straight teeth; and subsp. gallicus has both fixed diaspores with entire valves and long dispersible diaspores with toothed valves. In some populations of subsp. gallicus, plants can also have fruits at the base of the stems and such populations have been referred to var. subaegeus Maire in the W Mediterranean. However, these differences among subspecies are often less evident in the field and recognition of these taxa can be difficult due to the variability found in natural populations. In this study we used molecular markers to evaluate the infraspecific differentiation in R. bucephalophorus and to infer a phylogeographic scenario. As a framework for our initial studies, and to assist our collection of material, we used the infraspecific classification by Press (1988). Our study addresses the following questions: (1) Is R. bucephalophorus a monophyletic complex? (2) If so, is it possible to distinguish genetically different infraspecific taxa within this species? (3) Can we propose a phylogeny-based classification in R. bucephalophorus? and (4) Can we elucidate a coherent phylogeography of R. bucephalophorus? We used two approaches to answer these questions: the sequencing of nuclear ribosomal regions (internal transcribed spacers, nrITS), and a more variable DNA fingerprint method (amplified fragment length polymorphism, AFLP). ITS has been used successfully in the study of several old Tertiary lineages, such

as Anthyllis montana (Kropf et al., 2002), the Campanula lusitanica complex (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 different clades reported by Navajas-Pérez et al. (2005) in their phylogeny of the genus Rumex. If the clade including R. bucephalophorus is as old as estimated by Navajas-Pérez et al. (2005), ITS sequences could be a useful tool to distinguish genetically well-defined taxa. In contrast, AFLP markers are highly polymorphic and have been used successfully in differentiating among infraspecific taxa (Terrab et al., 2008) or closely related species (Tremetsberger et al., 2004; Ortiz et al., 2009; Balao et al., 2010). Moreover, AFLP markers are among the most commonly used in phylogeographic studies in recent years (Beheregaray, 2008). 2. Material and methods 2.1. Plant Material and DNA isolation We focused our sampling in the western area of distribution of R. bucephalophorus since this is where the four subspecies recognised by Press (1988) occur. A total of 204 individuals were sampled from 20 populations from N Africa (Atlantic and Mediterranean Morocco and Algeria), the Iberian Peninsula (Atlantic and SW coastlands and some inland localities), and Macaronesia (Table 1, Fig. 2). Employing the morphological criteria for subspecies adopted by Press (1988), these samples included 25 plants from 3 populations of subsp. bucephalophorus, 39 from 4 populations of subsp. canariensis (one of which, pop 7, belonged to var. fruticescens), 53 from 5 populations of subsp. hispanicus, and 87 from 8 populations of subsp. gallicus (41 individuals from 4 populations of 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 individuals per population, and dried in silica gel until processed for DNA isolation. Vouchers are deposited in the Herbarium of the University of Seville, Spain (SEV). Total genomic DNA, for nrDNA ITS and AFLP analyses, was extracted following the CTAB protocol (Doyle and Doyle, 1987) with modifications (Terrab et al., 2007). All sampled population extracts were checked for the presence and amount of DNA on 1% TAE agarose gels. 2.2. nrDNA ITS We randomly chose two individuals per population to analyse the nuclear ribosomal Internal Transcribed Spacer regions ITS1 and ITS2; to do that we used universal primers ITS4 and ITS5 (White et al., 1990). ITS1 and ITS2 were amplified using the same methodology as in Casimiro-Soriguer et al. (2010). We encountered a number of problems in sequencing ITS from Rumex. Among the 40 initial samples, 20 presented multiple rDNA arrays probably

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Table 1 Population-based estimates of genetic diversity and diaspore types in Rumex bucephalophorus. Total no. of fragments (Fragtot); % polymorphic fragments (Fragpoly); average gene diversity (HD); private fragments (Fragpriv), values given in brackets if they are fixed; rare fragments index (DW). Averages for subspecies are shown in bold. Asterisks indicate sequenced samples for ITS variation. Diaspore type: BD = buried diaspore, FD = fixed diaspore, SD = short-dispersible diaspore, LD = long-dispersible diaspore. Vouchers for each of these populations are deposited at SEV. The numbered locations in the table are also plotted in map on the Fig. 2. Taxa/localities a subsp. bucephalophorus 1. Algeria. Skikda. Kef Fatima 2. Spain. Málaga. Marbella 3. Spain. Cádiz. La Línea⁄

Diaspore type

Coordinates

Collector no

36°540 /7°020 36°290 /4°460 36°140 /5°190

SD, LD SD, LD FD, SD

EV: 242/07 MT: 363/06 MT: 10/07

b subsp. canariensis (Steinh.) Rech. fil. b1 var. canariensis 4. Madeira. Porto Santo island 5. Madeira I. Seixal⁄ 6. Madeira II. Garajau⁄

FD, SD FD, SD FD, SD

33°05 /16°18 32°480 /17°030 32°380 /16°510

MS:195/07 MS:196/07 MS: 194/07

b2 var. fruticescens (Bornm.) J.R. Press 7. Madeira III. Pico Arieiro⁄

FD, SD

32°440 /16°560

MS:285/07

0

HD

Fragpriv

DW

FST

5 11 9

100 120 119

90.0 82.5 87.3

0.10 0.08 0.09

1 2 5

7.5 28.0 24.0

8.3

113

86.6

0.09

2.7

19.8

11 7 11

144 99 126

81.2 66.7 76.2

0.10 0.06 0.08

7(1) 3 4

28.2 12.8 23.1

10

118

78.8

0.08

3

18.8

9.7

121.7

75.7

0.08

4.2

20.7

12 7 12 10 12

190 178 187 180 181

93.7 89.9 93.0 93.9 94.5

0.15 0.16 0.14 0.15 0.14

6 1 1 1 0

27.1 11.2 18.5 17.1 14.1

10.6

183.2

93.0

0.15

1.8

17.6

0.21 FD, SD FD, SD FD, SD FD, LD BD, FD, LD

43°330 /7°110 42°170 /8°500 42°160 /8°450 41°430 /8°520 39°200 /9°210

ST: 306/06 SC: 287/07 SC: 288/07 ST: 267/06 ER: 224/07

Mean

0.31 0.22 41°160 /3°480 40°570 /5°400 37°360 /3°080 35°110 /3°580

FD, LD FD, LD FD,LD FD, LD

MM: 225/07 ER: 223/07 JP: 221/07 ST: 61/07

Mean d2. var. subaegeus Maire 17. Spain. Huelva. Acebrón 18. Spain. Sevilla. Paradas⁄ 19. Morocco. Larache 20. Morocco. Kenitra⁄ Mean

Fragpoly

0.53 0

Mean

d subsp. gallicus (Steinh.) Rech. fil. d1. var. gallicus 13. Spain. Segovia. Villar de Sobrepeña 14. Spain. Salamanca. Pte de la Salud 15. Spain. Granada. Hoya de Guadix 16. Morocco. Alhucemas

Fragtot

0.30

Mean

c subsp. hispanicus (Steinh.) Rech. fil. 8. Spain. Lugo. San Cosme⁄ 9. Spain. Pontevedra I. El Tojar 10. Spain. Pontevedra II. Moaña⁄ 11. Portugal. Minho. Viana do Castello 12. Portugal. Estremadura. Peniche

N

8 9 12 12

115 95 181 163

96.5 93.7 95.0 98.2

0.10 0.08 0.14 0.13

0 0 1 3

4.3 4.4 18.8 17.9

10.2

138.5

95.8

0.11

1

11.3

12 11 12 11 11.5

126 122 182 174 151

94.4 89.3 90.1 87.4 90.3

0.10 0.09 0.14 0.12 0.11

0 1 2 0 0.7

9.3 7.8 20.8 16.1 13.5

10.9

144.7

93.1

0.11

0.9

12.4

0.26 BD, BD, BD, BD,

FD, FD, FD, FD,

LD LD LD LD

37°070 /6°310 37°160 /5°270 35°080 /6°080 34°130 /6°340

Mean

MT: MT: MT: MT:

4/07 13/07 158/07 145/07

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

as consequence of incomplete concerted evolution. In another 12, only 200 base pairs (bp) were amplified, likely due to diverse causes, such as presence of pseudogenes or secondary structure (Álvarez and Wendel, 2003; Nieto Feliner and Roselló, 2007). As a consequence, we obtained complete ITS1 and ITS2 sequences for only 8 of the 40 individuals. Fortunately, these successful samples 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. bucephalophorus (Genbank Nos. HQ389207–HQ389222). The aligned data matrix are deposited in the TreeBASE database (Accession No. S11620). We constructed a phylogeny using our ITS sequences along with GenBank accessions of related species belonging to the other three subgenera of Rumex. We selected species from the different clades of Rumex reported by Navajas-Pérez et al. (2005). Specifically, we included one species from subgenus Acetosella, the dioecious Rumex graminifolius (accession number: AJ831539, AJ844277); three species from subgenus Acetosa, the dioecious Rumex acetosa (AJ580774, AJ580790), the hermaphrodite–polygamous Rumex induratus (AJ580778, AJ580794) and the polygamous–gynodioecious Rumex hastatus (AF338218); and three hermaphrodite species 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-Pérez et al. (2005). To construct the phylogeny we used maximum parsimony analysis (PAUP ver. 4.0 beta 10), and full heuristic search option (10,000 replicates) to calculate bootstrapping values (Swofford, 2002). We estimated divergence times from the phylogeny, either within R. bucephalophorus or from its ancestors, using the Bayesian relaxed molecular clock method implemented in BEAST 1.4.7 (Drummond and Rambaut, 2007). Because Rumex belongs to a clade with a poor fossil record, the clock was calibrated by a ‘‘uniform prior’’ on two nodes previously estimated by Navajas-Pérez et al. (2005): 16–15 Mya for the appearance of dioecy in Rumex (Fig. 3, node A), and 13–12 Mya as the divergence time between the dioecious species of the subgenera Acetosella (R. graminifolius) and Acetosa (R. acetosa) (Fig. 3, node B). Furthermore, the hermaphrodite, polygamous or gynodioecious species of subgenera Acetosa (R. hastatus and R. induratus) and Rumex (R. conglomeratus, R. japonicus and R. aquitanicus) were included within the ingroup, while F. convolvulus was used to root the tree. We also assumed uncorrelation, Yule model for speciation, and automatic tuning of operators.

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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.

Analyses were run for 100 million generations (sampling every 1000th), with a burn-in of one million generations.

available at http://hordeum.oscs.montana.edu/genographer) using the ‘thumbnail’ option. Results were summarised as a matrix of presence/absence.

2.3. AFLPs

2.3.1. AFLP-based genetic diversity and population differentiation Total number of fragments (Fragtot), percentage of polymorphic fragments (Fragpoly) as well as the private fragment number (Fragpriv), were determined for each population, using FAMD version 1.08 (Schlüter and Harris, 2006). Mean genetic diversity (HD) was computed with Arlequin version 3.01 (Excoffier et al., 2005). The Rarity Index (equivalent to the frequency of down-weighted marker values; i.e., DW) was also calculated with AFLPdat (Ehrich, 2006) and R Statistical Software ver. 2.8.1 (R Development Core Team). The extent of genetic differentiation, measured as FST (the fixation index) as well as shared exclusive fragments (Fragpriv) was determined both for pairs of populations and taxa using Arlequin 3.01 (Excoffier et al., 2005). The relationship between pairwise FST values and geographical distances was investigated with Mantel tests based on Spearman rank correlations with 1 million permutations (package ade4 in R Statistical Software; Dray and Dufour, 2007).

All 204 sampled individuals were included in this study. Following the protocols established by Vos et al. (1995), genomic DNA (0.5 lg on average) was digested with two restriction endonucleases (EcoRI and MseI), the fragments ligated to doublestranded adaptors (EcoRI and MseI) at 37 °C for 2 h, then diluted 20-fold with TE0.1 buffer. Fragments with matching nucleotides were amplified (downstream of the restriction sites) using preselective primers based on EcoRI and MseI adaptors. This resulted in a 16-fold decrease in the number of fragments amplified. Preselective and selective amplifications were performed in a thermal cycler (Gene AmpÒ PCR system 9700, PE Applied Biosystems). In order to assess the reliability of the method, a random fraction (N = 22, i.e., 10.8%) of the samples was analysed twice from the first PCR. It was found that duplicate analyses were largely indistinguishable, 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 three primers that generated the most polymorphic fragments were selected. These were: MseI-CAC/EcoRI-AGC, MseI-CAT/EcoRI-ACG, MseI-CAG/EcoRI-ACT. Fluorescence-labelled selective amplification products were separated on a 3130xl Genetic Analyzers (Applied Biosystems) capillary sequencer with an internal size standard (GeneScan-500 ROX, PE Applied Biosystems). Alignment of the raw data with the size standard was performed with ABI Prism GeneScan software 2.1 (PE Applied Biosystems). AFLP fragments were scored for bands 80–500 bp in length with software developed at Montana State University (Genographer ver. 1.6.0,

2.3.2. Plant clustering Among-plant genetic distances (Nei and Li, 1979) were calculated from the matrix of AFLP scores and represented graphically with an unrooted neighbour-joining dendrogram built with SPLITSTREE 4.6 (Huson and Bryant, 2006). The bootstrap support for each branch was calculated with PAUP ver. 4.0 beta 10 (Swofford, 2002; neighbour-joining clustering, 10,000 replicates). We applied a principal co-ordinate analysis (PCoA) of a Euclidean matrix of similarities among individuals to investigate genetic distances and relationships between individuals. The PCoA was

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Fig. 3. Bayesian chronogram based on a maximum parsimony tree obtained from nuclear rDNA sequences (ITS1, ITS2) of Rumex bucephalophorus, and close relatives; Fallopia convolvulus 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).

performed with the program FAMD version 1.08 (Schlüter and Harris, 2006) and plotted with SPSS 15.0. Genetic relationships among populations were further explored with a Bayesian assignment technique implemented in STRUCTURE 2.2.3 (Pritchard et al., 2000; Falush et al., 2003) and the analysis was carried out at the freely available Bioportal (http://www.bioportal.uio.no/). This approach uses individual multilocus genotypes to construct clusters of genetically similar plants, in a way that maximises Hardy Weinberg equilibrium and minimises linkage disequilibrium within clusters. Following Falush et al. (2003) and Evanno et al. (2005) we used the ‘admixture’ and the ‘correlated allele frequency’ models, a combination judged appropriate when the aim is to infer subtle population structures, as expected in our dataset. 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 was determined from analyses that had varying values of K, from 1 to a maximum of 20 (i.e., the total number of populations). Ten independent runs were performed and averaged for each value of K. Furthermore, the partitioning (within and among populations and taxa) of genetic variation was determined with an Analysis of Molecular Variance (AMOVA; Excoffier et al., 2005) performed over all the studied samples. The effect on genetic variation of subspecies was also studied with AMOVA.

3. Results 3.1. Diaspore distribution In R. bucephalophorus subsp. bucephalophorus fixed non-dispersible diaspores (FD), long-dispersible diaspores (LD) and short-dispersible diaspores (SD) were found, but buried diaspores (BD) were always absent (Table 1). In subsp. canariensis both fixed and short-dispersible diaspores were recorded, in subsp. gallicus buried, 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 presents two pairs of teeth. In the other three subspecies internal bracts can be entire or toothed; when toothed, in subsp. canariensis they 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 ranged from 384 to 387 base pairs (bp). Maximum parsimony analysis generated 2 equally parsimonious trees that were similar, but in one the R. bucephalophorus complex is most closely related to the R. hastatus–R. induratus clade (without bootstrap support), and in

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population (Fragpriv), and the highest values of rare fragment index (DW) were found in populations of subsp. canariensis and bucephalophorus (Table 1). Only one population (pop. 4 of subsp. canariensis) showed one fixed private fragment, i.e., this fragment was found in all the individuals of the population. The highest fixation index value was found in subsp. canariensis (FST = 0.53) and the lowest in subsp. hispanicus (FST = 0.21; Table 1). The genetic dissimilarity between any two populations (as FST) significantly increased with increasing spatial distance (Mantel test, r = 0.518, N = 20, p = 0.002). The pairwise FST distances and the exclusive shared fragments (Table 2) agreed with the results mentioned above, showing that most populations diverged markedly 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, 11 and 12; Table 2). High values of FST were found between subsp. bucephalophorus and subsp. canariensis and also the other subspecies (0.48–0.35; Table 3). In contrast, the lowest FST value (0.15) was found between subsp. hispanicus and subsp. gallicus (Table 3). Interestingly, the two latter subspecies shared a markedly high number of pairwise private fragments (Fragpriv = 79, Table 3), and subsp. gallicus shared a considerable number of pairwise private fragments with the other subspecies (20 and 24, Table 3). In contrast, subsp. hispanicus and subsp. canariensis only shared three private fragments. The highest number of private fragments was found in subsp. canariensis (39, Table 3). When considering the two varieties of subsp. gallicus, the FST value between both varieties (0.20) is very similar to the values between subsp. hispanicus and these varieties (0.18 with var. gallicus and 0.19 with var. subaegeus; data not shown). In addition, the number of pairwise private fragments shared by both varieties (11) is less than those between subsp. hispanicus and these varieties (18 with var. gallicus and 13 with var. subaegeus; data not shown).

the other (shown in Fig. 3) it appears most closely related to the R. acetosa–R. graminifolius clade (also without bootstrap support). The length of the tree shown in Fig. 3 was 375 steps, the consistency index (CI) excluding autopomorphies was 0.80, and the retention index (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 supported monophyletic clade (98% Bootstrap, BS). All samples of R. bucephalophorus form a robust monophyletic group (99% BS) in which subsp. bucephalophorus is sister to all other subspecies, but with a very low support (51% BS, 11 nucleotide changes), whilst subsp. canariensis forms a strongly supported clade (81% BS), differing from the subsp. hispanicus plus subsp. gallicus 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. bucephalophorus began to diversify around 4.2 (95% HPD: 8.4–1.1) Mya into two phylogenetic lineages, one comprising R. bucephalophorus subsp. bucephalophorus, and the other the ancestral taxon of the three other subspecies (Fig. 3). Subspecies canariensis diverged at 0.7 Mya (95% HPD: 1.8–0.1) and var. fruticescens (a perennial) at 0.3 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 unambiguously scoreable DNA fragments were detected, of which 428 (97.9%) were polymorphic. The number of fragments for each primer combination was MseI-CAC/EcoRI-AGC: 132, MseI-CAT/EcoRIACG: 141, and MseI-CAG/EcoRI-ACT: 164. This number of fragments was sufficient to distinguish all 204 individuals as separate phenotypes. The populations of subsp. hispanicus presented the highest mean values of genetic diversity (HD = 0.15), those of subsp. gallicus showed intermediate values (HD = 0.11) and the populations of subsp. bucephalophorus and subsp. canariensis presented the lowest values (HD = 0.09 and 0.08, respectively; Table 1). The highest percentage of polymorphic fragments was found in subsp. hispanicus and gallicus (93%) and the lowest in subsp. canariensis (75.7%; Table 1). The highest numbers of private fragments, exclusive to each

3.4. AFLP-based plant clustering The neighbour-joining unrooted dendrogram of AFLPs resolved subsp. bucephalophorus as a separate cluster with 100% BS (Fig. 4). In the other large cluster, composed of the other three subspecies,

Table 2 Matrix of pairwise fixation index (AMOVA-derived FST) and pairwise shared private fragments (Fragpriv, above diagonal) for the 20 Rumex bucephalophorus populations. Extreme FST values are in bold, and empty cells denote that the number of shared fragments equals zero.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a

Subsp. bucephalophorus

Subsp. canariensis

1 – 0.37 0.26 0.60 0.66 0.62 0.63 0.48 0.46 0.47 0.45 0.44 0.37 0.45 0.36 0.35 0.48 0.50 0.45 0.49

Subsp. hispanicus

2

3

4

5

6

7a

– 0.28 0.69 0.73 0.71 0.71 0.60 0.59 0.59 0.60 0.58 0.57 0.61 0.53 0.53 0.61 0.63 0.58 0.61

9 – 0.65 0.70 0.68 0.67 0.56 0.55 0.55 0.55 0.54 0.50 0.56 0.47 0.48 0.57 0.59 0.55 0.58

1 – 0.53 0.45 0.54 0.58 0.57 0.57 0.55 0.54 0.56 0.61 0.49 0.53 0.61 0.61 0.57 0.60

2 – 0.58 0.58 0.57 0.56 0.55 0.54 0.51 0.56 0.61 0.49 0.50 0.61 0.61 0.56 0.60

3

2 1 3 – 0.59 0.59 0.59 0.58 0.55 0.56 0.62 0.51 0.51 0.62 0.62 0.57 0.60

Var. fruticescens.

– 0.52 0.61 0.60 0.60 0.58 0.56 0.59 0.63 0.52 0.55 0.62 0.64 0.59 0.62

8

9

10

11

12

Subsp. gallicus var. gallicus

Subsp. gallicus var. subaegeus

13

17 1

18

19 1

1 – 0.22 0.30 0.36

2

1

– 0.28 0.32

– 0.05

14

15

16

1 2

1

20

1 1 – 0.22 0.28 0.29 0.28 0.35 0.39 0.34 0.37 0.40 0.41 0.38 0.41

1 – 0.09 0.16 0.15 0.33 0.37 0.31 0.33 0.35 0.34 0.31 0.35

2 – 0.19 0.15 0.33 0.38 0.32 0.35 0.36 0.36 0.33 0.38

2 1

1 1

– 0.21 0.33 0.35 0.33 0.35 0.36 0.36 0.33 0.36

– 0.26 0.33 0.24 0.27 0.33 0.30 0.30 0.33

2

– 0.19 0.14 0.19 0.32 0.31 0.34 0.39

– 0.28 0.31 0.34 0.33 0.36 0.41

1

1

– 0.18 0.35 0.33 0.33 0.36

1 – 0.38 0.36 0.35 0.38

1

6 –

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

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

Subsp. bucephalophorus

Subsp. canariensis

Subsp. hispanicus

Subsp. gallicus

– 0.48 0.42 0.35 25

5 – 0.37 0.32 39

5 3 – 0.15 25

20 24 79 – 28

subsp. canariensis (99% BS) and subsp. hispanicus (76% BS) were also strongly supported, but subsp. gallicus was not a cohesive group (Fig. 4). The Principal Coordinate analysis (PCoA), based on Euclidean similarity matrix, showed that subsp. bucephalophorus and subsp. canariensis constituted separate and well distinguishable units (Fig. 5). The other two subspecies were clustered much closer together, but there was a trend to distinguish the plants into three groups: subsp. hispanicus, subsp. gallicus var. subaegeus and subsp. gallicus var. gallicus (Fig. 5). In fact, a Bayesian analysis conducted with STRUCTURE showed that K = 3 or K = 5 were the most likely number 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. gallicus joined together, while with K = 5, populations of the subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus appeared as distinct groups. Furthermore, this Bayesian analysis assigned the majority of individuals to their populations or groups of populations with a high probability. The results of analyses of molecular variance (AMOVA) showed that when no groups were considered, differences among plants accounted for 52.7% of variation (Table 4a). When the populations were grouped, the model considering three groups (subsp. bucephalophorus, subsp. canariensis, and subsp. gallicus + hispanicus), explained the largest fraction of variation (30.87%, Table 4b), followed by that considering five groups (subsp. bucephalophorus, subsp. canariensis, subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus) which explained 28.66% (Table 4d) of variation. The model considering four groups (the four subspecies) explained a lower percentage of variation (Table 4c).

4. Discussion 4.1. Diaspore distribution The peduncle and the position of the diaspore of R. bucephalophorus determine its ultimate function. Diaspores with thick peduncles (buried and fixed) remain attached to the plant until senescence and thus, they do not disperse. In contrast, diaspores with very thin peduncles (long- and short-dispersible) are easily detached from the plant and are dispersed by wind or epizoochory (Talavera, 2011). These four diaspore types can also differ in the morphology of the bracts (differing in number of teeth and in bract length) (López González, 1990). Thus, we can differentiate diaspores according to their function and/or morphology. All these diaspore traits have been traditionally used to distinguish infraspecific taxa in R. bucephalophorus (Press, 1988; López González, 1990). However, we have found that subsp. bucephalophorus can produce up to three functional diaspore types (fixed non-dispersible, long-dispersible and short-dispersible), subsp. gallicus can produce buried, fixed and long-dispersible diaspores, and the remaining 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 in another study showed that these diaspores can be found in some populations of this taxon (Talavera, 2011). However, neither populations nor plants of each subspecies simultaneously present all these 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. In the remaining taxa there is an overlapping number of teeth on the bracts and so this trait cannot be used to distinguish these taxa. Thus, our results clearly show that separation of all the infraspecific taxa of R. bucephalophorus cannot rely on these traits.

4.2. nrDNA ITS phylogeny Although ITS sequences were obtained for only eight of the 20 populations of R. bucephalophorus studied, the phylogenetic structure was consistent with data from AFLP markers for 204 individuals across all 20 populations. ITS analyses clearly show the monophyly of the clade comprising the Acetosa–Acetosella–Platypodium subgenera (Fig. 3, 98% BS). Given that the majority of species in this clade are currently distributed around the Mediterranean Basin, we can assume that R. bucephalophorus originated in this area at 15–16 Mya during the Miocene (Navajas-Pérez et al., 2005) although its infraspecific diversification began much later (Fig. 3). According to our relaxed molecular clock analysis, R. bucephalophorus began to diversify around 4.2 Mya into two phylogenetic lineages comprising R. bucephalophorus subsp. bucephalophorus, and the ancestor of the other three subspecies. From the group formed by the other taxa, only subsp. canariensis is robustly supported and estimated to have diverged during the Pleistocene, at about 0.7 Mya. Thus, results from ITS clearly show that subsp. bucephalophorus and subsp. canariensis are well differentiated taxa, whereas subsp. gallicus and subsp. hispanicus constitute a single group (see Fig. 3). The R. bucephalophorus lineage is estimated to have originated about 15 Mya, with extant taxa diversifing around 4 Mya (Fig. 3). During this time, the western margin of the embryonic Mediterranean had a very complicated geological history (Rosenbaum et al., 2002) with shifting islands, a reduced SE Iberian land area, and the Betic and Rifian sea corridors that gave access to the Atlantic. The latter closed between 7.16 and 6 Mya (Krijgsman et al., 1999a; Seidenkrantz et al., 2000), thus causing the gradual desiccation of the Mediterranean Basin (7.2–5.3 Mya) named the Messinian Salinity Crisis (Hsü et al., 1973; Bocquet et al., 1978; Krijgsman et al., 1999b; Duggen et al., 2003), and leading to marked climatic changes (Rouchy and Caruso, 2006; Murphy et al., 2009). Our data indicate that towards the end of this complicated period, the first diversification of R. bucephalophorus took place, with subsp. bucephalophorus as sister to a clade with the ancestor of the remaining subspecies. Thus, our results are in accordance with other phylogenetic studies of animals (García-París and Jockusch, 1999; Sanmartín, 2003; Fromhage et al., 2004; Martínez-Solano et al., 2004) and plants (Caujapé-Castells and Jansen, 2003; Hampe

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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), if higher than 50%, are indicated at each node. For details of populations, see Table 1.

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 expansion and diversification.

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Fig. 5. Ordination of AFLP data of Rumex bucephalophorus. Principal Coordinate Analysis (PCoA) based on a matrix of Euclidean similarity among the 204 studied individuals.

4.3. AFLP-based genetic diversity, population differentiation, and plant clustering The early divergence between subsp. bucephalophorus and the other subspecies is also supported by AFLP analyses that show that the genetic distance between this subspecies and the other subspecies 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-

667

guished morphologically as the diaspores are consistently large and with wide triangular teeth (López González, 1990). Moreover, the two populations of subsp. bucephalophorus from the Iberian Peninsula indeed have high DW values (pops. 2 and 3, Table 1), strongly suggesting they are old (Stehlik, 2002; Schönswetter and Tribsch, 2005). In contrast, the population of subsp. bucephalophorus from Algeria (pop. 1, Table 1) is among the most genetically impoverished of the species, suggesting that this is a recently established population, but it could also reflect diverse stochastic processes, such as genetic drift or bottlenecks (Ellstrand and Elam, 1993). The subspecies gallicus and hispanicus recognised by Press (1988) were not strongly supported by the molecular markers employed here. The maximum parsimony tree generated by the ITS data and the NJ dendrogram generated by the AFLP data indicate that the group of populations analysed from R. bucephalophorus fall into three groups, two of which coincide with subspecies bucephalophorus and canariensis, but the third group includes populations assigned to subsp. gallicus and subsp. hispanicus. However, the distinct clustering analyses, especially PCoA and STRUCTURE (Figs. 5 and 6; respectively), show that within this third group, three subgroups of populations can be distinguished. These subgroups correspond to the populations that were assigned to subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus, which differ in geographical distribution and diaspore traits (Press, 1988). It is notable that populations of subsp. hispanicus, subsp. gallicus var. subaegeus and subsp. gallicus var. gallicus showed similar genetic distances among them (Table 2). This suggests that the three subgroups should be considered at the same taxonomic level, but since they are not as clearly separated as subsp. bucephalophorus and subsp. canariensis, we think that it is better 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 genetic diversity and high indices of rare fragments (Table 1); these facts point towards an ancient origin of these populations (Bonin

Fig. 6. Genetic structure of Rumex bucephalophorus inferred by Bayesian clustering of AFLP data. Assignment of 204 individuals into K genetically distinguishable groups. Each individual is represented by a vertical bar coloured according to the assigned group(s). The 20 populations are identified following Table 1. The upper and lower bands show the most stable and likely assignments estimated by structure (at K = 3 and K = 5) (see text).

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Table 4 Results 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 three different ways (b–d). All models were highly significant (P < 0.00001). Grouping of populations and source of variation

Df

Variance component

Variation (%)

a Global analysis Among populations Among plants

FST (95%CI)

19 184

22.15 24.68

47.30 52.70

b [1–3][4–7][8–20] Among groups Among populations Among plants

2 17 184

17.07 13.55 24.68

30.87 24.50 44.63

0.31 (0.28–0.34)

c [1–3][4–7][8–12][13–20] Among groups Among populations Among plants

3 16 184

14.02 11.81 24.68

27.76 23.38 48.86

0.28 (0.24–0.30)

d [1–3][4–7][8–12][13–16][17–20] Among groups Among populations Among plants

4 15 184

14.09 10.41 24.68

28.66 21.16 50.18

0.29 (0.26–0.31)

0.47 (0.45–0.49)

et al., 2007). The exceptions are pops. 13 and 14 from the Spanish Central Plateau of subsp. gallicus var. gallicus, and pops. 17 and 18 from the Guadalquivir valley (SW Iberian Peninsula) of subsp. gallicus var. subaegeus. Given that coastal or sub-coastal zones are the main habitats for the hispanicus–gallicus group, it is likely that the inland populations are more recently founded. The Central Plateau populations appear to be derived from Betic and Rifean populations (pops. 15 and 16, Fig. 2) because, as shown by our results (Table 2), they are genetically very close, with markedly low genetic distances (FST) between both groups of populations (pops. 13 and 14 vs. 15 and 16; Table 2). The low genetic diversity (HD, Fragpoly, and DW; Table 1) of Central Plateau populations of subsp. gallicus var. gallicus (pops. 13 and 14) suggests a very recent origin derived from southern populations (pops. 15 and 16). The south to north migration of the plants of Spain has been recently documented in other species such as Ferula loscosii (Pérez-Collazos et al., 2009). The Guadalquivir valley populations of subsp. gallicus var. subaegeus (pops. 17 and 18, Fig. 2) probably originated from the populations of subsp gallicus var. subaegeus of NW Morocco (pops. 19 and 20, Fig. 2) since they are genetically similar (Fig. 4) and NW Morocco populations have higher genetic diversity than Guadalquivir valley populations (Table 1). In fact, there are numerous examples of species that crossed from the Atlantic coast of N Africa to the Iberian 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 supported by AFLP data as it has a very high number of private fragments (39, Table 3) and its genetic distance to the other taxa is also high (FST = 0.32–0.48, Table 3). According to our results (Fig. 3), subsp. canariensis diverged recently (0.73 Mya), presumably as a consequence of the arrival on Macaronesia of immigrants from continental populations during the early Pleistocene. The fact that subsp. canariensis and subsp. gallicus share the highest number of pairwise private fragments (Table 3), and have the lowest FST value (Table 3), indicate that subsp. canariensis is most closely related to subsp. gallicus. On the other hand, populations of subsp. canariensis are very diverse and genetic distances among them are very high. Reproductive isolation of populations is common in species that occur on oceanic volcanic islands where the immigrants underwent a strong adaptive radiation (Sanmartín et al., 2008). This could explain the strong genetic differences between the population of Porto Santo Island and the three populations of Madeira Island (Table 2). However, the three populations of Madeira Island also showed markedly high genetic distances (Table 2). Multiple

colonisation events on Madeira Island could explain these differences. Moreover, the orography of the island is very rugged and these populations although geographically close could also be reproductively isolated. Two populations (5 and 6) are on opposite sides of the island separated by a high mountain range and the third population (pop. 7) is in the high part of this range. The latter consists of perennial woody plants and is the only locality where R. bucephalophorus exhibits this habit. This supports the idea that these populations of Madeira are isolated, because gene exchange between them would have eliminated or spread the perennial habit. These perennial woody plants were previously treated as subsp. fruticescens (Rechinger, 1939), and subsequently recognised as a variety of subsp. canariensis (Press, 1988). Both annual and suffruticose species occur in the genus Rumex, but it is unusual for both habits to appear in the same species. There is ample suggestion based on morphology of the genus Rumex to support the view that the annual habit is usually derived from perennial (Löve and Kapoor, 1967). However, according to our molecular clock analyses, var. fruticescens diverged only some 0.32 Mya (Fig. 3), thus suggesting that in R. bucephalophorus the suffruticose habit evolved from the annual condition. This pattern has been also reported in numerous molecular phylogenetic plant studies in oceanic 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 and Hart, 1996; Panero et al., 1999; Thiv et al., 1999; Helfgott et al., 2000; Barber et al., 2002; Mansion et al., 2009). In fact, the reversion from annual to perennial seems to be genetically simple, as demonstrated in Arabidopsis thaliana (Melzer et al., 2008). In conclusion, despite the fact that R. bucephalophorus originated in the Miocene, its subspecific diversification is likely to have coincided with the end of Messinian Salinity Crisis; this supports the evidence that this geological period played an important role in the diversification of many plant species. The two molecular markers we employed indicate that R. bucephalophorus is a monophyletic species, and also clearly differentiate subsp. bucephalophorus as sister to all other subspecies, and subsp. canariensis as the most recently derived subspecific taxon; subsp. hispanicus and subsp. gallicus constitute a monophyletic group, within which three subgroups can be recognised that correspond to subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus. Given that, at present, these subgroups are not clearly separated, neither genetically nor morphologically, we propose that it would be better to consider all three as varieties of subsp. gallicus.

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