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Journal of Biogeography (J. Biogeogr.) (2009) 36, 1384–1397

SPECIAL ISSUE

Phylogeographic patterns in Hypochaeris section Hypochaeris (Asteraceae, Lactuceae) of the western Mediterranean Marıá Ańgeles Ortiz1*, Karin Tremetsberger1,2, Tod F. Stuessy2, Anass Terrab1,2, Juan L. Garcıá-Castanõ1 and Salvador Talavera1

1

Departamento de Biologıá Vegetal y Ecologıá, Universidad de Sevilla, Sevilla, Spain and 2 Department of Systematic and Evolutionary Botany, Faculty Centre Biodiversity, University of Vienna, Vienna, Austria

ABSTRACT

Aim To analyse phylogeographic patterns in the four species of Hypochaeris sect. Hypochaeris, evaluating possible areas of origin and the microevolutionary processes that have shaped their morphology, genetics and distribution. Location Western Mediterranean area. Methods We applied amplified fragment length polymorphism (AFLP) markers to a total of 494 individuals belonging to 82 populations of Hypochaeris arachnoidea, H. glabra, H. radicata and H. salzmanniana to determine population structure. Results Populations with the largest proportion of private and rare AFLP fragments were found in Morocco. This region was consequently inferred to be the ancestral area for H. arachnoidea, H. glabra, H. radicata and H. salzmanniana. The Guadalquivir River (southern Spain) was inferred to be an effective dispersal barrier for H. glabra and H. radicata. The Strait of Gibraltar was inferred to be a somewhat weaker barrier than the Guadalquivir River for H. radicata and a much weaker barrier for H. glabra. The main barrier for H. salzmanniana coincides with the extension of the Rif Mountains to the Atlantic coast in Morocco, and the Strait of Gibraltar is a much weaker barrier for this species. Hypochaeris arachnoidea appears to have originated in the Atlas Mountains.

*Correspondence: Marıá Ańgeles Ortiz, Departamento de Biologıá Vegetal y Ecologıá, Universidad de Sevilla, Apdo-1095, 41080 Sevilla, Spain. E-mail: [email protected]

Main conclusions The highest levels of genetic variation in La Mamora forest (H. glabra and H. salzmanniana) or the adjacent central Middle Atlas (H. arachnoidea and H. radicata) in Morocco suggest that these areas were a centre of origin of Hypochaeris sect. Hypochaeris. All three potential barriers – the Guadalquivir River, the Strait of Gibraltar, and the Rif Mountains – have been important in shaping genetic diversity in species of section Hypochaeris. Keywords AFLP, Atlas Mountains, Guadalquivir River, Iberian Peninsula, phylogeography, population genetic variation, rare fragments, Rif Mountains, Strait of Gibraltar.

INTRODUCTION The western Mediterranean region has undergone dramatic geomorphological and environmental changes during the past 8 Myr (Thompson, 2003). The principal events were the closure of the connection to the Atlantic (7–5.33 Ma) and the opening of the Strait of Gibraltar (5.33 Ma). The pre-Mediterranean sea had two seaway connections with the Atlantic Ocean: the Betic and the Riffian corridors. The Betic Corridor became the future Guadalquivir valley, 1384

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which now separates the old Palaeozoic lands of the Sierra Morena to the north from the Tertiary lands of the Betic Sierras; the Riffian Corridor occupied the modern Loukos and Sebou river valleys in Morocco, which separate the Rif Mountains to the north from the Atlas ranges to the south. These sea channels constituted formidable barriers for the migration of plants and animals between North Africa and Europe. This situation changed dramatically with the closure of the two Mediterranean–Atlantic channels (at 7–5.33 Ma), creating the so-called Messinian Salinity Crisis, with the drying ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd

Phylogeographic patterns in Hypochaeris section Hypochaeris out of much of the Mediterranean Basin, followed by the opening of the Strait of Gibraltar (at around 5.33 Ma). Until c. 3.2 Ma, the climate of the south-western Mediterranean area seems to have been subtropical, although cold and arid conditions were apparently established in North Africa from around 3.9 Ma (Estabrook, 2001). A mediterranean-type climate, with warm, wet winters and hot, dry summers, seems to have become established during the Pliocene (around 3.5 Ma), before the onset of the Northern Hemisphere glaciations at around 2.4 Ma. The latter gave rise to oscillating changes in sea level in the Strait of Gibraltar area that exposed and submerged islands in this area during the glacial cycles. Up to now, relatively few studies have been concerned with the evolutionary and biogeographical consequences of Pleistocene glacial cycles on northern African and southern European taxa. There were three important biogeographical barriers for terrestrial plant species in the western Mediterranean area after the Messinian age: the Guadalquivir valley, the Strait of Gibraltar, and the Loukos and Sebou valleys in Morocco. The most important bioclimatic changes in the Mediterranean Basin over the last 5 Myr were: (1) the establishment of the mediterranean climate during the Pliocene (c. 3.5 Ma), and (2) the glacial periods of the Pliocene and Pleistocene. The onset of the mediterranean climate caused very dramatic changes in the woodlands, particularly in the understorey, with selective pressure to shorten plant life cycles, or to modify underground rhizomes to form corms. As a consequence, more than 50% of the plant species of the Mediterranean Basin are annuals (Talavera, 1991), and rhizomatous plants are confined to the more humid environments (e.g. streams, lagoons and springs) or to the mesic understorey. The Quaternary glacial periods shaped the expansion and diversification of plant populations along the western Mediterranean. During glacial periods, the sea level was lowered, for example by 130 m in the Last Glacial Maximum, some 20,000 years ago, and the temporarily emergent coastlines occupied by plant populations were used as land-bridges for colonization. During interglacials, the sea transgressed and coastal populations suffered drastic reductions and possibly extinctions. All of these influences have led to complex evolutionary and biogeographical patterns in the biota of the Mediterranean region, especially in the western part (Jong, 1998; Ve´la & Benhouhou, 2007). Further assessment of the impact of these environmental changes in the western Mediterranean requires the study of additional groups, such as the genus Hypochaeris, which can serve as a model system. The genus consists of c. 58 species world-wide, with only 15 confined to the Mediterranean region, three in Eurasia, and more than 40 in South America. Of those in the Mediterranean area, section Hypochaeris, with four species, is centred in the western Mediterranean. This section is monophyletic (Tremetsberger et al., 2005) and diversified between c. 1.7 and 2.0 Ma (K. Tremetsberger et al., unpublished data). In general, Hypochaeris is a suitable genus with which to evaluate biogeographical patterns because of the many previous studies conducted on its cytology and cytogenetics (Cerbah et al., 1995; Ruas et al.,

1995, 2008; Weiss et al., 2003; Weiss-Schneeweiss et al., 2003, 2007, 2008), DNA sequences (Cerbah et al., 1998; Samuel et al., 2003; Tremetsberger et al., 2005), AFLP population analyses (Stuessy et al., 2003; Tremetsberger et al., 2003a,b, 2004, 2006; Muellner et al., 2005; Mraz et al., 2007; Ortiz et al., 2007, 2008) and reproductive biology (Ortiz et al., 2006). To date, South American species have been the focus of research because they represent the greatest concentration of species diversity. We review available data for Hypochaeris sect. Hypochaeris and focus on four biogeographical issues: (1) the possible area of origin of this section; (2) the impact of the Strait of Gibraltar on populations of H. glabra, H. radicata and H. salzmanniana; (3) the impact of the Guadalquivir River in southern Spain on population divergence in H. glabra and H. radicata; and (4) patterns of genetic divergence in H. arachnoidea in Morocco. MATERIALS AND METHODS Hypochaeris sect. Hypochaeris Section Hypochaeris is a monophyletic group (Tremetsberger et al., 2005) composed of four species: H. arachnoidea Poir., H. glabra L., H. radicata L. and H. salzmanniana DC. In addition to having morphological differences, these species differ in a range of other parameters: (1) life-form: H. radicata is perennial whereas H. arachnoidea, H. glabra and H. salzmanniana are annuals; (2) somatic chromosome number: H. glabra has 2n = 10 chromosomes, whereas H. arachnoidea, H. radicata and H. salzmanniana have 2n = 8 chromosomes (Tremetsberger et al., 2005); and (3) distribution: H. glabra and H. radicata are widespread in the Mediterranean region and also occur as weeds world-wide, whereas H. arachnoidea is endemic to North West Africa (Morocco and Algeria) and H. salzmanniana is restricted to the Atlantic coast of Morocco and south-western Spain (Ca´diz). The natural habitat of the former three species is the understorey of open Quercus woodland (although weedy invasive populations of H. glabra and H. radicata occur in different habitats), but H. salzmanniana occurs principally on coastal dunes. All four species can hybridize in the greenhouse, but in nature hybrids have never been found in the natural range of the species. However, Parker (1975) found sterile hybrids between H. glabra and H. radicata in England, where both species are non-native. As with many composites, Hypochaeris species have sporophytic self-incompatibility (SSI), requiring obligatory cross-pollination (usually mediated by solitary bees) for fruit-set. Within section Hypochaeris, SSI is found in H. arachnoidea and H. radicata and in most populations of H. salzmanniana, whereas H. glabra is self-compatible (Ortiz et al., 2006). Sampled populations We sampled a total of 494 individuals belonging to 82 populations of the four species of section Hypochaeris (see

Journal of Biogeography 36, 1384–1397 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd

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M. A´. Ortiz et al. Appendix S1 in Supporting Information). Most populations were in the western Mediterranean region (the centre of diversity of the group – see below), but we also included other populations from throughout the distributional ranges: 15 populations of H. glabra (five from Morocco, G1–G5; eight from the Iberian Peninsula, G6–G13; one from the Canary Islands, G14; and one from Chile, G15); 44 populations of H. radicata [11 from Morocco, R1–R11; five from the Central Mediterranean area (Italy, Sicily and Tunisia), R12–R17; 17 from the Iberian Peninsula, R18–R34; three from West and Central Europe (France, the Netherlands and Austria), R35– R37; three from Asia (Taiwan and South Korea), R38–R40; and four from South America (Argentina and Chile), R41– R44]; nine Moroccan populations of H. arachnoidea (A1–A9); and 14 populations of H. salzmanniana (six from Morocco, S1–S6; and eight from Ca´diz, Spain, S7–S14). Of these, all populations of H. arachnoidea, and Moroccan and central and south-eastern Spanish populations of H. glabra were newly analysed in this study. Populations of H. radicata are those of Tremetsberger et al. (2004) and Ortiz et al. (2008), and populations of H. salzmanniana are those of Tremetsberger et al. (2004) and Ortiz et al. (2007). The number of individuals sampled in each population is shown in Appendix S1. Fresh leaves of the plants were collected at least 1 m apart and dried in silica gel. Vouchers of all sampled populations were deposited in the Herbarium of the University of Seville (SEV, Spain) and/or the University of Vienna (WU, Austria). DNA isolation and AFLP analysis Total genomic DNA was extracted from dry leaf material following the CTAB (cetyl trimethyl ammonium bromide) protocol (Doyle & Doyle, 1987) with modifications. The amplified fragment length polymorphism (AFLP) procedure followed established protocols (Vos et al., 1995) with modifications (Tremetsberger et al., 2003a, 2004; Ortiz et al., 2007). The six primer combinations for the selective polymerase chain reaction (PCR) selected by Tremetsberger et al. (2004) were applied to all four species: MseI-CTCG/EcoRI-ATC (Fam), MseI-CAC/EcoRI-ACG (Hex), MseI-CTA/EcoRI-ACC (Ned), MseI-CTG/EcoRI-ACA (Fam), MseI-CTC/EcoRI-AGG (Hex) and MseI-CTGA/EcoRI-AAC (Ned). In addition, three more primers selected by Ortiz et al. (2007), namely MseI-CAC/ EcoRI-ACT (FAM), MseI-CTC/EcoRI-ATC (HEX) and MseICTG/EcoRI-AAC (NED), were used to obtain better resolution in H. salzmanniana. The fluorescence-labelled selective amplification products were separated on a 5% polyacrylamide gel with an internal size standard (GeneScan-500 ROX; PE Applied Biosystems, Foster City, CA, USA) on an automated sequencer (ABI 377; Perkin-Elmer, Waltham, MA, USA). Amplified fragments of 60–500 bp were scored and exported as a presence/absence matrix using ABI Prism genescan analysis Software 2.1 (PE Applied Biosystems) and genographer (ver. 1.6.0 Montana State University 2001; available at: http://hordeum.oscs.montana.edu/genographer/). The error rate, based on phenotypic comparisons among the 37 1386

replicated individuals of H. radicata (Bonin et al., 2004), amounted to 1.12% (Ortiz et al., 2008). Data analyses AFLP markers can be used to infer phylogenetic relationships based on measures of genetic distance for closely related species (Beismann et al., 1997; Mueller & Wolfenbarger, 1999; Zhang et al., 2001; Despre´s et al., 2003). This method was successful in determining phylogenetic relationships among South American species of Hypochaeris (Tremetsberger et al., 2006). For this purpose we scored one individual from five populations of each species, covering as much as possible of the natural distributional range (H. glabra: G4, G5, G8, G10 and G12; H. radicata: R7, R21, R26, R27 and R28; H. arachnoidea: A1, A4, A6, A7 and A9; H. salzmanniana: S3, S6, S9, S11 and S13). From this presence/absence matrix, we constructed a dendrogram using the neighbour-joining (NJ) method in conjunction with Nei & Li’s (1979) genetic distances in paup* (ver. 4.0b10; Sinauer Associates, Sunderland, MA, USA). Support for each node was tested by 10,000 bootstrap replicates. We used famd ver. 1.1 (Schlu¨ter & Harris, 2006) to interchange between different file formats and calculate the proportion of private AFLP fragments (i.e. those confined to only one species; Fpriv). Pairwise exclusive shared fragments (i.e. fragments exclusively shared by a pair of species that are not present in any other species; Fsh) and pairwise fixation indices based on Euclidean distances [analysis of molecular variance (AMOVA)-derived FST values; arlequin ver. 3.01 (Excoffier et al., 2005)] were assessed for each species. For each species independently, the AMOVA-derived population pairwise FST matrix, based on the squared Euclidean distances, was calculated with arlequin ver. 3.01 (Excoffier et al., 2005) and imported into splitstree ver. 4.6 (Huson & Bryant, 2006) so as to construct a NJ population dendrogram. As a measure of within-population diversity, we assessed the percentage of polymorphic fragments (Fpoly), as well as the number of private fragments (Fpriv, confined to one population or group of populations) for all populations of the four species of Hypochaeris sect. Hypochaeris. We also assessed the number of fragments that were shared exclusively between pairs or groups of populations (Fsh), and we calculated another index of diversity DW (‘frequency-down-weighted marker values’) with AFLPdat (Ehrich, 2006). Population values were estimated by making a table with the presence of markers by population, dividing each marker by the total number of occurrences of this marker in the dataset, and summing these relative values to give the rarity index for this particular population (rarity 2). Private and rare fragments accumulate through time and are, therefore, a measure of population antiquity (Stehlik et al., 2002; Scho¨nswetter & Tribsch, 2005). As another measure of genetic variability, we also calculated the average gene diversity (HD; arlequin ver. 3.01; Excoffier et al., 2005).


Phylogeographic patterns in Hypochaeris section Hypochaeris In order to examine the population structure of the four species, H. arachnoidea, H. glabra, H. radicata and H. salzmanniana, we conducted an approach based on statistical inference with Bayesian clustering methods using baps ver. 5.1 (Corander et al., 2003, 2004; Corander & Marttinen, 2006; available at http://www.abo.fi/fak/mnf/mate/jc/software/baps. html), which uses stochastic optimization instead of Markov chain Monte Carlo (MCMC) to find the optimal partition. The simulation was run from K = 2 to K = N + 1, where N is the number of populations analysed in each species, except for H. radicata, for which the simulation was run to 20, with five replicates for each K. We used the option ‘clustering of individuals’ to estimate the admixture coefficients for the reference individuals, and this was performed with the following settings: minimal size of clusters at four individuals, 100 iterations to estimate the admixture coefficients for the individuals, 200 simulated reference individuals from each population, and 20 iterations. A post hoc Tukey–Kramer honestly significant difference (HSD) test was applied to detect differences in private fragments and DW among populations. We considered differences significant at a 5% confidence level (Bonferroni correction applied). We used AMOVA (arlequin ver. 3.01; Excoffier et al., 2005) to distribute genetic variation into portions assignable to differences between predefined hierarchical groups (FCT), among populations within these groups (FSC), and among populations across the entire study area (FST) (Turner et al., 2000). We tested with AMOVA for possible effects of potentially major geographic barriers (the Strait of Gibraltar, Guadalquivir River, and extension of the Rif Mountains) for the three species H. glabra, H. radicata and H. salzmanniana. FST values were calculated with arlequin ver. 3.01 (based on squared Euclidean distances), and, in order to counterbalance unequal sample sizes, qST values [based on samples of two individuals, i.e. qST(2)] were calculated according to the rarefaction method (Hurlbert, 1971; Mousadik & Petit, 1996). RESULTS Phylogenetic relationships among the four species of section Hypochaeris The AFLP primer combinations applied to five populations (one individual per population) of each of the four species of section Hypochaeris generated 32–118 fragments, of which a high percentage (72.5–100%) were polymorphic. The total number of fragments was 428, of which 406 (94.8%) were polymorphic. The NJ dendrogram with the four species of section Hypochaeris (Fig. 1) had the same topology as the rps16 intron tree (Tremetsberger et al., 2005): H. glabra is sister to H. arachnoidea, H. radicata and H. salzmanniana. Each of the four species is well supported, with bootstrap support (BS) ranging from 89% (for H. radicata) to 100% (for H. glabra and H. salzmanniana). Hypochaeris arachnoidea is sister to

H. salzmanniana (95% BS) and H. radicata is sister to this latter group. The species with the largest number of private fragments (76, of which 10 were fixed) was H. salzmanniana (see Table 1). Hypochaeris glabra exhibited the fewest private fragments (52), but the highest number of fixed private fragments (20). Hypochaeris radicata shared most exclusive fragments with the other three species (a total of 45), and H. glabra had the lowest number of exclusive fragments shared with other species (a total of 26). Most exclusive fragments were shared between H. salzmanniana and H. arachnoidea (20), indicating a close relationship. AMOVA-derived FST values indicated a weaker relationship between H. glabra and H. salzmanniana (FST = 0.67). Hypochaeris glabra The six AFLP primer combinations applied to H. glabra yielded a total of 242 fragments, of which 81.8% were polymorphic. The unrooted NJ dendrogram and Bayesian analysis, conducted with baps, for the 15 populations of H. glabra (Fig. 2a), showed three main clusters: the first (70% BS) includes the populations from Morocco (populations G1–G5) and the Spanish populations from areas south of the Guadalquivir River (G6–G7); the second cluster includes populations from central and south-eastern Spain (G8–G10; 100% BS); and the remaining populations form a distinct Bayesian cluster (but without NJ bootstrap support) that groups the populations from Donãna and south-western Sierra Morena (southwestern Spain, G11–G13) together with those from the Canary Islands (G14) and Chile (G15; the latter two supported by 75% BS). Thus, the main splits within this species were between the populations from Morocco and from the south of the Guadalquivir River in Spain, those from Donãna, from the south-western Sierra Morena, and from the extraEuropean introduced accessions (Canary Islands and Chile), and those from central and south-eastern Spain. Genetic diversity measures for each population of H. glabra are shown in Table 2. The highest numbers of polymorphisms and average gene diversity were in the Moroccan populations (%Fpoly = 28.61 and HD = 0.091) and the central and south-eastern Spanish populations (%Fpoly = 28.55 and HD = 0.091; Fig. 2b). Moroccan populations had the highest number of rare fragments and private fragments (DW = 18.0 and Fpriv = 4.8), and the lowest numbers were in Spanish populations south of the Guadalquivir River (DW = 6.3 and Fpriv = 0.0). Hypochaeris radicata The population structure of this species was analysed in detail by Ortiz et al. (2008). These data were used to construct a NJ unrooted tree and perform Bayesian clustering (Fig. 3a). The main clusters were: Morocco (R1–R11); south of the Guadalquivir (Spain; R18–R24); Donãna (Spain; R25–R26); the


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M. A´. Ortiz et al.

Figure 1 Neighbour-joining (NJ) dendrogram of 20 individuals of Hypochaeris arachnoidea, H. glabra, H. radicata and H. salzmanniana analysed for AFLP and using Nei & Li’s genetic distances. Bootstrap values based on 10,000 permutations are indicated at each node (if > 50%).

Table 1 Pairwise exclusive shared fragments (Fsh; values below diagonal), pairwise fixation index (AMOVA-derived FST; above diagonal), and private fragments (Fpriv) applied to five populations (one individual per population) of each species of Hypochaeris sect. Hypochaeris. The values are based on analysis of a total of 428 AFLP fragments. Numbers in parentheses refer to fixed fragments. FST Fsh

H.glabra H.radicata H.salzmanniana H.arachnoidea

H. glabra H. radicata H. salzmanniana H. arachnoidea Fpriv

– 13 (1) 5 8 52 (20)

0.56 – 18 14 64 (3)

0.67 0.44 – 20 (2) 76 (10)

0.60 0.34 0.45 – 62 (4)

Central Mediterranean (R12–R17); and a cluster including south-western Sierra Morena, northern, central and eastern Spain, and all the introduced accessions of the species (R27– R44). The averaged genetic diversity parameters are shown in Fig. 3b. Morocco is seen to be the group with the highest diversity. 1388

Hypochaeris salzmanniana The population structure of this coastal species was analysed in detail by Ortiz et al. (2007). The results of the unrooted NJ dendrogram (Fig. 4a) showed five main clusters: Algeciras Bay (Spain; S13–S14), Sierra San Bartolome´ (Spain; S11–S12), south of the extension of the Rif Mountains to the Atlantic coast in Morocco coincident with south of the Loukos River (S3–S6), north of the extension of the Rif Mountains (Morocco; S1–S2), and Barbate (Spain; S7–S10); the latter two grouped in the same Bayesian cluster. The averaged genetic diversity parameters are shown in Fig. 4b. The population from south of the Loukos River in Morocco had the highest diversity. Hypochaeris arachnoidea The six AFLP primer combinations applied to H. arachnoidea yielded a total of 499 fragments, of which 93.2% were polymorphic. This species presented four main clusters (Fig. 5a), the first from Taza, in the Rif Mountains (A1; 100% BS), the second from Essaouira, on the southern


Phylogeographic patterns in Hypochaeris section Hypochaeris

(b)

(a)

Figure 2 Sampling localities and results of AFLP analysis of Hypochaeris glabra: (a) neighbour-joining (NJ) unrooted dendrograms, based on analysis of molecular variance (AMOVA)-derived FST values (scale bar indicates an FST value of 0.1). Bootstrap values based on 10,000 permutations are indicated at each node (if > 50%). (b) Sampling localities of H. glabra in the western Mediterranean region. The bars indicate the mean number ± SE of the private fragments (Fp; max–min: 4.8–0), the rare fragments index (DW; max–min: 18.0–6.3) and the genetic diversity (HD; max–min: 0.091–0.034). Colour-coding of populations indicates results of Bayesian clustering (baps).

Moroccan Atlantic coast (A2–A4; 60% BS), and the third from the Anti Atlas Mountains (A5–A6; 70% BS). The last Bayesian cluster (not a group in the NJ tree) comprises geographically dispersed populations from Nador, on the Mediterranean coast of Morocco (A7), Tiznit, in the foothills of the Anti Atlas Mountains, close to the Atlantic coast (A8), and Tizi-N-Test from the High Atlas Mountains (A9). Genetic diversity measures for each population of H. arachnoidea are shown in Table 2. The highest number of polymorphisms, the highest average gene diversity, and the highest number of rare fragments were found in the Anti Atlas populations (A5–A6; %Fpoly = 71.24; HD = 0.150; DW = 53.65; Fig. 5b). The highest number of private fragments was found in Taza (A1; Fpriv = 24). Evaluation of biogeographical hypotheses The analyses of molecular variance between populations on either side of the potential biogeographical barriers (Strait of Gibraltar, Guadalquivir River and extension of the Rif

Mountains; Table 3) show that all three potential barriers may have been important in shaping genetic diversity in species of section Hypochaeris. For H. glabra and H. radicata, the main breaks coincide with the Guadalquivir River in Andalusia and the Strait of Gibraltar. The Guadalquivir River accounts for 39.1% of the variance in H. glabra (compared with 28.2% across the Strait of Gibraltar). For H. radicata, the Guadalquivir River and the Strait of Gibraltar account for 18.4% and 16.0% of genetic variance, respectively. For H. salzmanniana, the Strait of Gibraltar accounts for very little variance (11.9%), whereas the extension of the Rif Mountains to the Atlantic coast in Morocco accounts for 17.6% of the variance. The qST pairwise values correspond with the AMOVAderived FST values (data not shown). A post hoc Tukey–Kramer HSD test found significant differences in the number of private fragments and DW between populations from Morocco and all other populations (in H. glabra and H. radicata) and between populations from south of Loukos River and all other populations (in H. salzmanniana).


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M. A´. Ortiz et al. Table 2 Total number of fragments (Ftot), percentage polymorphic fragments (Fpoly), private fragments (Fpriv), rare fragments (DW), average gene diversity (HD) and number of samples (NAFLP) in populations of Hypochaeris glabra (G1–G15) and H. arachnoidea (A1–A9) sampled for AFLP. Species/Pop. H. glabra G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 H. arachnoidea A1 A2 A3 A4 A5 A6 A7 A8 A9

Localities

Ftot

Fpoly (%)

Fpriv

DW

HD

Morocco, Tanger Asilah Larache Kenitra, La Mamora 1 Kenitra, La Mamora 2 S Guadalquivir, Spain, Barbate Punta Paloma C & SE Spain, Caćeres Jaen, Santa Elena Almerıá, Tabernas SW Sierra Morena, Spain, Valverde Donãna, Spain, Hinojos P.N. Donãna Introduced, Spain. Canary Islands ˜ uble Chile, N

141 133 162 167 145 112 119 145 142 142 126 119 129 125 124

22.69 10.53 40.74 40.12 28.96 – 10.08 12.41 47.18 26.06 3.17 5.04 15.50 6.40 10.48

2 1 5 12 (1) 4 0 0 0 5 0 0 0 2 1 (1) 0

13.7 11.2 20.9 27.2 17.1 5.7 6.8 14.0 18.1 14.6 10.4 8.0 11.1 10.2 9.0

0.0677 0.0306 0.1339 0.1298 0.0939 – 0.0489 0.0326 0.1404 0.1007 0.0163 0.0245 0.0816 0.0326 0.0354

5 4 5 5 5 2 2 5 5 3 2 2 2 2 3

Taza Essaouira, Moulay-Bouzerktour Essaouira J. Amsittene Anti Atlas, Biougra Tafraoute Nador, High Atlas, Anti Atlas, Nador Tifnit Tizi-N-Test

160 198 197 217 230 240 190 224 235

60.62 67.17 64.97 66.36 69.56 72.92 60.00 70.09 73.62

24 (2) 8 4 12 5 23 9 17 20

40.4 34.4 31.9 45.6 44.8 62.5 34.2 50.0 54.1

0.0836 0.1106 0.1049 0.1140 0.1549 0.1441 0.0998 0.1331 0.1516

9 10 10 10 7 10 9 10 9

DISCUSSION Morocco: the ancestral area for section Hypochaeris Morocco has been inferred as the ancestral area of H. radicata and H. salzmanniana based on the presence of many private and rare AFLP fragments in this region (Ortiz et al., 2007, 2008). In the case of H. radicata, the greatest numbers of private and rare fragments occur in the Rif, where it is found in wet pastures associated with Quercus suber forests, and in the central Middle Atlas, where it grows in wet pastures associated with forests of Cedrus atlantica. Ortiz et al. (2007) inferred H. salzmanniana to have originated in the southern part of its distributional range in Morocco, namely in the once more extensive Q. suber forests of the Sebou valley, which occur today as remnant vegetation in La Mamora forest, close to Kenitra in the north-western foothills of the Middle Atlas. Hypochaeris glabra also exhibits its highest genetic diversity in the La Mamora area (G3–G5), where the species occurs in the understorey of the Q. suber forest, in mixed populations with H. salzmanniana. The Sebou valley seems to have played an important role in diversification within the section. This diversification might be related to arid–wet cycles starting 2.3 Ma (Suc, 1984). However, we do not know the effects on 1390

NAFLP

ancestral variation of more recent climatic events (such as glacial cycles). Hypochaeris arachnoidea is distributed throughout the Atlas Mountains in Morocco and Algeria in dry, open woodland (Oberprieler, 2002; Oberprieler & Vogt, 2002; Fo¨rther & Podlech, 2003). Its probable origin is in the Atlas Mountains, although the exact location is difficult to determine, mainly because of limited sampling (populations from Algeria and parts of the Moroccan Middle Atlas have not been collected). Moreover, unlike the case for the other three species of section Hypochaeris, there is no clear geographical genetic pattern. Bayesian clustering groups several very distant populations, and we interpret this as being the result of dispersal over long distances, possibly associated with human migrations. Mountain populations of H. arachnoidea (A1, A4–A6, A9) have higher mean levels of private and rare fragments (DW) than do populations closer to the sea (A2–A3, A7–A8; 16.8 vs. 9.5 and 49.5 vs. 37.6), and this pattern is also seen in populations around Essaouira (A4 vs. A2–A3). We hypothesize, therefore, that H. arachnoidea originated in a mountain habitat, and that the Atlas Mountains, at a lower elevation, served as a Pleistocene refugium. The Atlas Mountains are known for their high level of endemism (Que´zel, 1978; Fennane & Ibn Tattou, 1998), also evident in other species of Hypochaeris, for



(b)

(a)

Figure 3 Sampling localities and results of AFLP analysis of Hypochaeris radicata: (a) neighbour-joining (NJ) unrooted dendrograms, based on analysis of molecular variance (AMOVA)-derived FST values (scale bar indicates an FST value of 0.1). Bootstrap values based on 10,000 permutations are indicated at each node (if > 50%). (b) Sampling localities of H. radicata in the western Mediterranean region. The bars indicate the mean number ± SE of the private fragments (Fp; max–min: 4.1–0.5), the rare fragments index (DW; max–min: 9.4–4.8) and the genetic diversity (HD; max–min: 0.091–0.050). Colour-coding of populations indicates results of Bayesian clustering (baps).

example H. angustifolia and H. leontodontoides (Galań de Mera & Vicente Orellana, 1998a,b). Phylogeographic patterns in H. glabra, H. radicata and H. salzmanianna Hypochaeris glabra and H. radicata show very similar biogeographical patterns of genetic diversity. However, in H. glabra these results must be considered with caution because unequal sample sizes were analysed. Based on high numbers of private and rare fragments, we infer a Moroccan origin for H. glabra and H. radicata (Ortiz et al., 2008). From here, H. glabra appears to have first dispersed to the region north of the Guadalquivir River (south-western Sierra Morena, Donãna, central and south-eastern Spain) and then world-wide. The same basic pattern can be inferred for H. radicata (Ortiz et al., 2008). The second dispersal of both H. glabra and H. radicata from Morocco across the Strait of Gibraltar was to the region south of the Guadalquivir River, as evidenced by strong genetic similarity between Moroccan populations and Spanish populations south of the Guadalquivir River in both species (for more details, see Ortiz et al., 2008). The Donãna populations of H. radicata are isolated and restricted to humid zones close

to lagoons, and the two populations analysed (R25/El Corchuelo and R26/El Acebroń) are strongly genetically and morphologically divergent (Ortiz et al., 2006, 2008). This is not the case for H. glabra, which is widespread in the Donãna area. The similarity of populations of H. glabra and H. radicata on both sides of the Strait of Gibraltar cannot be linked to the closing of the Mediterranean Sea during the Messinian Salinity Crisis 6–7 Ma, because section Hypochaeris is estimated to be of only Pliocene or Pleistocene age [1.7–2.0 Ma (95% Bayesian highest posterior probability density interval = 0.6–3.5 Ma); Tremetsberger et al., 2005; K. Tremetsberger et al., unpublished data]. Hypochaeris salzmanniana is also hypothesized to have originated in southern Moroccan Quercus woodlands and migrated north (Ortiz et al., 2007). The main genetic division within the species is between populations on either side of the extension of the Rif Mountains to the Atlantic coast in northern Morocco. From northern Morocco, the taxon may have crossed the Strait of Gibraltar during the Pleistocene, when sea levels were lower (Ortiz et al., 2007). It developed differences in its mating compatibility system during northward migration, from completely self-incompatible individuals in all Moroccan and Algeciras Bay populations, through mixed


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M. A´. Ortiz et al. (b)

(a)

Figure 4 Sampling localities and results of AFLP analysis of Hypochaeris salzmanniana: (a) neighbour-joining (NJ) unrooted dendrograms, based on analysis of molecular variance (AMOVA)-derived FST values (scale bar indicates an FST value of 0.1). Bootstrap values based on 10,000 permutations are indicated at each node (if > 50%). (b) Sampling localities of H. salzmanniana in the western Mediterranean region. The bars indicate the mean number ± SE of the private fragments (Fp; max–min: 18.3–3.0), the rare fragments index (DW; max–min: 50.7–15.8) and the genetic diversity (HD; max–min: 0.146–0.044). Colour-coding of populations indicates results of Bayesian clustering (baps).

populations with self-compatible and self-incompatible individuals in Barbate, to self-compatible individuals in Sierra San Bartolome´ and Zahara (Ortiz et al., 2006, 2007). Pleistocene glacial impact on H. glabra, H. radicata and H. salzmanniana European and African landmasses were not directly connected by a continuous land bridge when sea levels were lower in the Pleistocene, but their coasts were considerably closer than they are today, especially on the Atlantic side of the Strait of Gibraltar, where the sea floor is not as deep as it is on the Mediterranean side (Pou, 1989; Yokoyama et al., 2000; Patarnello et al., 2007). Emergent islands present periodically during glacial periods in the Strait of Gibraltar area also favoured contact between the two continents (Collina-Girard, 2001). This periodical closeness of the African and European landmasses probably facilitated the colonization of H. glabra, H. radicata and H. salzmanniana from North Africa to the south-west of the Iberian Peninsula (Ortiz et al., 2007, 2008). The Strait of Gibraltar appears to be a rather strong barrier to gene flow in H. radicata and a weak one in H. glabra and 1392

H. salzmanniana. However, at least two dispersals from Morocco to the Iberian Peninsula are inferred for H. glabra and H. radicata (this paper and Ortiz et al., 2008). Hypochaeris salzmanniana is a coastal species growing on sand dunes along the beaches and might have been better adapted to conditions presented by the exposed sea floor during glacial periods than were H. glabra and H. radicata. The normal habitat of H. glabra is on sandy soils in woodlands, rather than coastal dunes. Hypochaeris radicata is confined to more humid habitats in the understorey of Mediterranean Quercus woodland. Impact of the Guadalquivir River on H. glabra and H. radicata Hypochaeris glabra and H. radicata share a principal genetic split across the Guadalquivir River (Spain) in the western Mediterranean region. In H. radicata, the morphologically and genetically divergent Donãna populations have an intermediate position between the two groups north and south of the Guadalquivir River. We hypothesize, on the basis of our results, that ancestral populations of H. glabra and H. radicata, like those of H. salzmanniana, expanded out of northern



(a)

(b)

Figure 5 Sampling localities and results of AFLP analysis of Hypochaeris arachnoidea: (a) neighbour-joining (NJ) unrooted dendrograms, based on analysis of molecular variance (AMOVA)-derived FST values (scale bar indicates an FST value of 0.1). Bootstrap values based on 10,000 permutations are indicated at each node (if > 50%). (b) Sampling localities of H. arachnoidea in the western Mediterranean region. The bars indicate the mean number ± SE of the private fragments (Fp; max–min: 24.0–8.0), the rare fragments index (DW; max–min: 46.2–37.3) and the genetic diversity (HD; max–min: 0.150–0.084). Colour-coding of populations indicates results of Bayesian clustering (baps).

Africa across the Strait of Gibraltar area into the southern Iberian Peninsula during the Quaternary (Ortiz et al., 2007). First, both species arrived north-west of the Guadalquivir River, probably because the coastline was more extensive and consequently closer to Morocco at that time. These populations expanded, corresponding to the modern populational systems in Donãna, Sierra Morena, northern, central and eastern Spain, and possibly Portugal. In a second more recent dispersal event, H. glabra and H. radicata reached a more easterly area of the Iberian Peninsula, giving rise to the southern Guadalquivir populations. Interestingly, the Guadalquivir River is still a modern barrier preventing the admixture of populations, as it is an important agricultural area in which H. glabra and especially H. radicata are seldom found. The Sierra Morena to the north-west of the Guadalquivir River and the Betic Cordillera to its south-east offer rather different soil conditions. The Sierra Morena consists

of acidic Precambrian and Palaeozoic terrain, whereas the Betic Cordillera is predominantly calcareous. In the Sierra Morena, H. radicata grows frequently and abundantly in the understorey of Quercus forests. In the rest of the Iberian Peninsula north of the Guadalquivir River, the species grows in anthropogenically modified sites on different substrates. Differentiation across the Guadalquivir Basin has been documented in several animal genera (Busack, 1986; GarcıáParı´s et al., 1998, 2003; Garcıá-Parı´s & Jockusch, 1999; Sanmartıń, 2003) and in two plant genera apart from Hypochaeris: Anthoxanthum (Pimentel et al., 2007) and Senecio (Comes & Abbott, 1998). In Discoglossus and Salamandra (Amphibia), it is thought that lineages were isolated by the opening of the Betic Strait or later as a result of the formation of the fluvial system during the Pliocene, and that this isolation has been maintained until recently by the Guadalquivir River Basin (Garcıá-Parı´s et al., 1998; Garcıá-Parı´s & Jockusch, 1999).


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M. A´. Ortiz et al. Table 3 Comparison of analyses of molecular variance (AMOVA) across three major geographic barriers, the Strait of Gibraltar, Guadalquivir River and Loukos River, for the three species Hypochaeris glabra, H. radicata and H. salzmanniana. Groupings of populations are shown in brackets. Source of variation

d.f.

Sum of squares

H. glabra [G1–G13] Populations 12 789.45 Individuals 34 349.32 Strait of Gibraltar [G1–G5], [G6–G12] Groups 1 251.72 Populations 11 537.73 Individuals 34 349.32 Guadalquivir River [G1–G7], [G8–G12] Groups 1 326.95 Populations 11 462.50 Individuals 34 349.32 H. radicata [R1–R34] Populations 33 3830.74 Individuals 129 2701.48 Strait of Gibraltar [R1–R17] [R18–R34] Groups 1 619.71 Populations 32 3211.03 Individuals 129 2701.48 Guadalquivir River [R1–R24] [R25–R34] Groups 1 669.53 Populations 32 3161.21 Individuals 129 2701.48 H. salzmanniana [S1–S14] Populations 13 2065.29 Individuals 126 3200.30 Strait of Gibraltar [S1–S8], [S9–S14] Groups 1 468.80 Populations 12 1507.88 Individuals 126 3200.30 Loukos River [S1–S10], [S11–S14] Groups 1 557.41 Populations 12 1507.88 Individuals 126 3200.30

Variance components

Variance (%)

F-values

95% confidence interval

15.54 10.27

60.21 39.79

FST = 0.602

8.36 10.96 10.27

28.23 37.05 34.71

FCT = 0.282 FSC = 0.516 FST = 0.653

0.22–0.32

12.39 9.00 10.27

39.13 28.42 32.45

FCT = 0.391 FSC = 0.467 FST = 0.675

0.32–0.44

19.85 20.94

48.67 51.33

FST = 0.487

7.17 16.58 20.94

16.04 37.10 46.86

FCT = 0.160 FSC = 0.442 FST = 0.531

0.11–0.20

8.41 16.25 20.94

18.44 35.64 45.92

FCT = 0.184 FSC = 0.437 FST = 0.541

0.13–0.22

13.35 25.40

34.45 65.55

FST = 0.344

4.90 10.76 25.40

11.93 26.22 61.86

FCT = 0.119 FSC = 0.298 FST = 0.381

0.08–0.15

7.56 10.03 25.40

17.58 23.33 59.09

FCT = 0.176 FSC = 0.283 FST = 0.409

0.13–0.21

ACKNOWLEDGEMENTS

REFERENCES

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Phylogeographic patterns in Hypochaeris section Hypochaeris Weiss-Schneeweiss, H., Tremetsberger, K., Schneeweiss, G.M., Parker, J.S. & Stuessy, T.F. (2008) Karyotype diversification and evolution in diploid and polyploid South American Hypochaeris (Asteraceae) inferred from rDNA localization and genetic fingerprint data. Annals of Botany, 101, 909–918. Yokoyama, Y., Lambeck, K., Deckker, P.D., Johnston, P. & Fifield, L.K. (2000) Timing of the Last Glacial Maximum from observed sea-level minima. Nature, 406, 713–716. Zhang, L.-B., Comes, H.P. & Kadereit, J.W. (2001) Phylogeny and Quaternary history of the European montane/alpine endemic Soldanella (Primulaceae) based on ITS and AFLP variation. American Journal of Botany, 88, 2331–2345.

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BIOSKETCH

SUPPORTING INFORMATION

The focus of the research team is on the study of the evolutionary history of the genus Hypochaeris in relation to other plant groups, combining molecular studies with studies of morphology and reproductive biology. S.T. and T.F.S. formulated the research questions; M.A´.O., K.T. and A.T. collected the data and prepared them for publication; J.L.G.-C. performed the statistical analyses; and M.A´.O. and K.T. led the writing.

Additional Supporting Information may be found in the online version of this article:

Editor: Christine Maggs

Appendix S1 Species, geographical–Bayesian groups, populations, localities, geographical coordinates, collector numbers and sample sizes for analysed populations of Hypochaeris arachnoidea, H. glabra, H. radicata and H. salzmanniana.

This paper stems from a contribution initially presented at the conference Origin and Evolution of Biota in Mediterranean Climate Zones: an Integrative Vision, held in Zurich on 14–15 July 2007.


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