Phylogeography above the species level for perennial species in a composite genus

1 Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. OPEN ACCESS – RESEARCH ARTICLE Phylogeography above the species level for perennial species in a composite genus Karin Tremetsberger1,2,*, María Ángeles Ortiz1, Anass Terrab1, Francisco Balao1, Ramón Casimiro-Soriguer1,3, María Talavera1 and Salvador Talavera1 1Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apdo. 1095, 41080 Seville, Spain. 2Present address: Institute of Botany, Department of Integrative Biology and Biodiversity Research, University of Natural Resources and Life Sciences, Gregor Mendel Straße 33, 1180 Vienna, Austria. 3Present address: Departamento de Biología, CASEM, Universidad de Cádiz, Campus Río San Pedro, 11510 Puerto Real, Spain. *Corresponding author AoB PLANTS Advance Access published December 7, 2015 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 2 Corresponding author’s e-mail address: [email protected] Running head: Phylogeography in the western Mediterranean Received: 22 April 2015; Revised: 30 July 2015; Accepted: 11 November 2015 ABSTRACT In phylogeography, DNA sequence and fingerprint data at the population level are used to infer evolutionary histories of species. Phylogeography above the species level is concerned with the genealogical aspects of divergent lineages. Here, we provide a phylogeographic study to examine the evolutionary history of a western Mediterranean composite, focusing on the perennial species of Helminthotheca (Asteraceae, Cichorieae). We used molecular markers (AFLP; ITS and plastid DNA sequences) to infer relationships among populations throughout the distributional range of the group. Interpretation is aided by biogeographic and molecular clock analyses. Four coherent entities are revealed by Bayesian mixture clustering of AFLP data, which correspond to taxa previously recognized at the rank of subspecies. The origin of the group was in western North Africa, from where it expanded across the Strait of Gibraltar to the Iberian Peninsula and across the Strait of Sicily to Sicily. Pleistocene lineage divergence is inferred within western North Africa as well as within the western Iberian region. The existence of the four entities as discrete evolutionary lineages suggests that they should be elevated to the rank of species, yielding H. aculeata, H. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 3 comosa, H. maroccana and H. spinosa, whereby the latter two necessitate new combinations. Keywords: Amplified fragment length polymorphism; Helminthotheca; Iberian Peninsula; phylogeography; Strait of Gibraltar; western Mediterranean region; western North Africa. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 4 INTRODUCTION Intraspecific phylogeography as originally defined by Avise et al. (1987) is concerned with the geographic distribution of genealogical lineages at the level of conspecific populations. Originally based upon animal mitochondrial DNA sequence data, phylogeographic studies today make use of a variety of molecular techniques and are based upon DNA sequence and fingerprint data from the mitochondrial, plastid and nuclear genomes. The field of phylogeography today extends from the level of conspecific populations to the supraspecific level (Avise 2000) and targets organisms from the bacteria and eukaryote domains of life as well as viruses. Phylogeography of closely related species spans the boundary between reticulate and divergent relationships, where a partially braided collection of allelic pathways of interbreeding individuals bifurcates into two such collections (Avise 2000). Likewise, genealogical data at the population level of closely related species are especially helpful for explaining distributions resulting from historical and ecological factors. A region that offered many opportunities for ecogeographic and effective geographic isolation (Sobel et al. 2010) due to its variegated geologic and climatic history since the Miocene is the western Mediterranean region. The formation of the Betic-Rif orogen led to the desiccation of the Mediterranean Sea between 6 and 5.3 million years ago (mya; Hsü et al. 1973), enabling the migration of animals and plants between the two continents (Bocquet et al. 1978). At the end of the Messinian, the Strait of Gibraltar opened and water from the Atlantic Ocean refilled the dry at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 5 Mediterranean Basin (Lonergan and White 1997), thus again disconnecting north-western Africa from south-western Europe. Major climatic events after the opening of the Strait of Gibraltar were the advent of the Mediterranean climate and Pleistocene climatic oscillations. In northern Africa, the appearance of a local or temporary dry season was already recognizable in the floristic composition of the Miocene and this climate trend was accentuated in the Pliocene (Quézel 1978). In the northwestern Mediterranean area, a Mediterranean climatic rhythm with summer drought appeared in the Pliocene, at approximately 3.2 mya (Suc 1984). In the Pleistocene, the water level of the Mediterranean Sea underwent great oscillations. During the last glacial maximum (0.02 mya), it was 120 m lower than today, so that the oceanic barrier between north-western Africa and south-western Europe was much reduced in extension, though not completely disrupted (Thiede 1978). The western Mediterranean region was an important refugium for temperate as well as Mediterranean plants and animals during the Pleistocene glacial periods (Médail and Diadema 2009). It is supposed that the changing environmental conditions and appearance/disappearance of biogeographic barriers have massively triggered plant and animal diversification in the Pliocene and Pleistocene (Fiz-Palacios and Valcárcel 2013). The genus Helminthotheca (Asteraceae, Cichorieae, Hypochaeridinae) has a primarily western Mediterranean distribution. It has been separated from Picris at the generic level on the basis of the conspicuously enlarged outer involucral bracts and comprises the annual, heterocarpic species H. echioides and H. balansae and the perennial, homocarpic species H. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 6 aculeata, H. comosa and H. glomerata (Picris comosa auct. alg.; Lack 1974, 1975). Helminthotheca aculeata has another subspecies, H. aculeata subsp. maroccana, in addition to the nominal subspecies. Similarly, H. comosa also has another subspecies, H. comosa subsp. lusitanica, in addition to the nominal subspecies. The most recent common ancestor (MRCA) of extant taxa of Helminthotheca can be traced back to the Pliocene or Pleistocene (Tremetsberger et al. 2013). It is thus likely that populations have been affected by climatic changes occurring in the Mediterranean region in this epoch, possibly leading to range shifts, disruptions of previously continuous ranges and secondary contacts. This study aims at shedding light on the evolutionary history of the perennial species of Helminthotheca. Using molecular makers from the nuclear and plastid genomes, we addressed the following questions: (1) Does genetic grouping correlate with the present taxonomic treatment? (2) Where did perennial species of Helminthotheca originate? (3) Along which routes did they spread to occupy their present areas? The inferred evolutionary history is discussed with respect to the evolutionary forces that might have been relevant for speciation. METHODS Plant material We sampled populations belonging to subspecies of Helminthotheca aculeata and H. comosa in Algeria, Italy (Sicily), Morocco, Portugal and Spain (Table 1, Fig. 1). Helminthotheca echioides was included as outgroup. Vouchers of all populations used are deposited in the herbaria SEV and/or WU. The Algerian species H. balansae and H. glomerata (Quézel and Santa at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 7 1963) could not be sampled for this study. In the field, leaves from individual plants were conserved in silica-gel. Distribution ranges of the taxa under study were compiled from survey of material in the herbaria at Barcelona (BC), Geneva (G), Madrid (MA), Montpellier (MPU), Paris (P), Salamanca (SALA) and Seville (SEV). Locations mentioned in Pignatti (1982) and Sauvage (1961) were also used. DIVA-GIS ver. 7.1.7.2 (Hijmans et al. 2001) was used to display distribution ranges and sample locations. AFLP The protocol for extraction of genomic DNA from silica-gel dried leaves, restriction-ligation (EcoRI and MseI), preselective and selective amplification, and electrophoresis on a capillary sequencer (3130xl Genetic Analyzer, Applied Biosystems) exactly followed Tremetsberger et al. (2009). Three selective primer combinations [EcoRI(Fam)-ACA/MseI-CAT, EcoRI(Vic)- AGG/MseI-CTC, and EcoRI(Ned)-AAC/MseI-CTT] were selected following a primer trial testing twelve selective primer combinations, based on the criteria of interpretability (presence/absence), spread of bands in the region 50-500 bp, and number of bands (approximately 70-90 per primer combination). Raw data were aligned with the GeneScan 500 ROX size standard using ABI PRISM GeneScan ver. 3.71 (© Applied Biosystems). Presence/absence of bands in all individuals and replicates was scored with Genographer ver. 1.6.0 (Benham 2001) after normalizing on total signal in one file. We scored 96 individuals from 25 populations of the perennial species of Helminthotheca. Twenty-two individuals were replicated from restriction-ligation and used to calculate the error rate as the ratio between observed number of phenotypic differences and total number of phenotypic comparisons between replicates at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 8 (Bonin et al. 2004). Three individuals of H. echioides were used as outgroup. Only loci observed in the ingroup were scored in the outgroup. We employed split decomposition (Bandelt and Dress 1992) for constructing a phylogenetic network, which is preferred over a strictly bifurcating tree at the population level, where gene flow is to be expected. FAMD ver. 1.108 (Schlüter and Harris 2006) was used to calculate a matrix of Nei and Li (1979) distances with the length of the restriction enzyme’s recognition sequence set to six between all pairs of individuals. This matrix was imported into SplitsTree ver. 4.8 (Huson and Bryant 2006) to construct a NeighborNet network. Splits, whose weight did not exceed a threshold of 0.001, were removed. Bayesian admixture clustering (Corander et al. 2003; Corander and Marttinen 2006) was used to estimate the number of genetically coherent clusters in the perennial species of Helminthotheca with BAPS ver. 5.2 (Corander et al. 2004, 2008). The maximum number of populations for mixture clustering was set to 20 (5×), 25 (5×), and 30 (5×). For admixture clustering based on results of mixture clustering, the minimum size of a population was set to 1, the number of iterations to 200, and the number of reference individuals to 200 with 20 iterations each. In order to determine which taxon might be ancestral, we generated a phylogenetic tree, which also included three individuals of the outgroup, H. echioides, by neighbour joining analysis based on Nei-Li distances in PAUP* ver. 4.0b10 (Swofford 2003). The support of nodes was estimated by 500 bootstrap replicates. Clusters containing only individuals of the same at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 9 population or taxon were collapsed using FigTree ver. 1.3.1 (Rambaut 2006- 2009). In order to distinguish between incongruent species trees, we adopted the approach of Bryant et al. (2012) to infer species trees from AFLP data in the framework of the multispecies coalescent, implemented in SNAPP ('SNP and AFLP Phylogenies'), a Bayesian MCMC sampler, which interfaces with the BEAST package (Drummond and Rambaut 2007). Nine individuals per taxon were included in the analysis: H. aculeata subsp. aculeata, A1-A5; H. aculeata subsp. maroccana, M1-M3; H. comosa subsp. comosa, C2-C6; and H. comosa subsp. lusitanica, S1-S3, S5 and S6. Individuals of putative hybrid origin (see below) were not included. We ran two separate Markov Chain Monte Carlo (MCMC) chains, each with 7 million states, without correction for dominance, in BEAST ver. 2.1.3 (Bouckaert et al. 2002-2014). Trees and associated parameter values were logged every 1,000 states. Results of the MCMC runs were analysed with Tracer ver. 1.5.0 (Rambaut and Drummond 2003-2005), discarding the first 10% as burn-in. Trees created in the two MCMC runs were combined with LogCombiner ver. 2.1.3, again discarding the first 10% of each run as burn-in. A species tree was drawn from the combined trees with DensiTree ver. 2.1.11 (Bouckaert 2010). Within-population genetic diversity was calculated as 'average gene diversity over loci' in Arlequin ver. 3.5.1.2 (Excoffier et al. 2005; only for populations with a minimum of three individuals). As indicators of divergence due to long-lasting isolation of populations, we estimated the number of private AFLP bands in a population (i.e., those bands occurring exclusively in the respective population) and the rarity index. Only populations with a at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 16 Network analysis (Fig. 2A) shows relationships among individuals of the four clusters. The major split is across the Strait of Gibraltar separating the H. comosa subsp. comosa/H. comosa subsp. lusitanica-cluster from the H. aculeata subsp. maroccana/H. aculeata subsp. aculeata-cluster. Within the Afro-Sicilian group, the H. aculeata subsp. maroccana-populations M1 and M2 from the Middle Atlas branch off basally, whereas the H. aculeata subsp. aculeata-populations (A1-A5) are derived (Fig. 2C). Within the Iberian group, the H. comosa subsp. comosa-populations (C2-C6) branch off basally, whereas the H. comosa subsp. lusitanica-populations (S1-S8) are derived. Evaluation of sampled trees from the coalescent approach to species tree reconstruction revealed an effective sample size (ESS) of > 10,000 for the two combined runs. The traces did not show signs of non-convergence. All clades have posterior probabilities of 100%, yielding a single consensus tree (Fig. 3A). In a tree lacking short branches, results of analyses with and without correction for dominance do not differ (Bryant et al. 2012), so that we relied only on the analysis without correction for dominance. The sequence of divergence suggested by the obtained species tree is: first, divergence of an Afro-Sicilian and an Afro-Iberian lineage; second, divergence of H. aculeata subsp. maroccana and H. aculeata subsp. aculeata in the Afro-Sicilian lineage; and third, divergence of H. comosa subsp. comosa and H. comosa subsp. lusitanica in the Afro-Iberian lineage. Accumulation of private and rare AFLP bands in populations due to mutations indicates long-term in-situ persistence of populations. This is in contrast to newly established populations, which should have fewer such bands. The mean number of private bands and the rarity index are slightly at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 17 higher in populations of the Afro-Sicilian lineage than in populations of the Afro-Iberian lineage (Table 2). This trend is accentuated, when the estimates are calculated for taxa rather than for populations. Helminthotheca aculeata subsp. aculeata (populations A1-A5 [Nindividuals = 11]) has 20 private bands and a rarity index of 4.1. Similarly, H. aculeata subsp. maroccana (populations M1-M3 [Nindividuals = 13]) has 19 private bands and a rarity index of 4.1. Helminthotheca comosa subsp. comosa (populations C3-C6 [Nindividuals = 18]) has 16 private bands and a rarity index of 2.6, and H. comosa subsp. lusitanica (populations S2, S5, S7, and S8 [Nindividuals = 20]) has 12 private bands and rarity index of 2.2. In parallel to estimates of divergence, withinpopulation genetic diversity is also slightly higher in populations of the Afro- Sicilian lineage than in populations of the Afro-Iberian lineage (Table 2). DNA sequences The G+C content of ITS1 and ITS2 sequences is very similar among accessions of the perennial species of Helminthotheca (mean = 53.3%; SD = 0.2%) and H. echioides (mean = 54.0%; SD = 0.3%), suggesting absence of pseudogenes. The ITS2 secondary structure is common to all individuals of Helminthotheca analysed [see SUPPORTING INFORMATION]. It is also well in accordance with the ITS2 secondary structure of Asteraceae presented by Goertzen et al. (2003). No compensatory base changes (Coleman 2007) exist among individuals of Helminthotheca, neither when comparisons are made among perennial species nor when these are compared with H. echioides. The ITS tree (Fig. 4A) reveals two groups. Sequences of populations M1-M3 are in basal position together with one sequence of population AM1 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 18 and one sequence of population CM. All remaining sequences are in a derived group with 72% bootstrap support. The plastid DNA tree (Fig. 4B) also reveals two main groups. One group with 79% bootstrap support contains sequences of the Afro-Sicilian AFLP group (all but two sequences of populations M1-M3 and all sequences of populations AM1, AM2, and A1-A5). The other group with 81% bootstrap support contains sequences of the Iberian AFLP group (populations C1-C6, CS and S1-S8) together with one sequence of population M1, one sequence of population M3 and sequences of population CM. Hybrids Differences in the individual trees derived from the different data sets allow identifying individuals of hybrid origin. Individuals of population CM largely correspond to the 'maroccana'-AFLP group (with two individuals showing admixture from the 'comosa'-AFLP group), one individual has the more basal 'maroccana'- and the other the more derived 'aculeata/comosa/lusitanica'-ribotype, but all individuals have the 'comosa/lusitanica'-plastid type. Similarly, one individual of population M1 and one individual of population M3 also have the 'comosa/lusitanica'-plastid type. One individual of population AM1 and both individuals of population AM2 morphologically resemble H. aculeata and have the 'aculeata/comosa/lusitanica'-ribotype, but correspond to the 'maroccana'- AFLP group. Finally, some individuals of population CS show evidence of admixture by Bayesian admixture clustering of AFLP data. When individuals of putative hybrid origin are excluded, Bayesian inference results in a bestat UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 19 scoring species tree derived from ITS and plastid DNA sequences (Fig. 3B) that is congruent with the species tree derived from AFLP data. Ancestral area and age estimation Model-based biogeographic reconstruction of ancestral areas (Fig. 5) gives an idea of the biogeographic history of the perennial species of Helminthotheca, albeit with little confidence. Western North Africa with its mountain ranges Rif and Middle Atlas is revealed as the ancestral area of the entire clade as well as of the common ancestor of H. aculeata subsp. aculeata and H. aculeata subsp. maroccana. The common ancestor of H. comosa subsp. comosa and H. comosa subsp. lusitanica is inferred to have lived in an area spanning W North Africa, the W Betic region, and the W Iberian region. Molecular clock analysis reveals that the minimum age of the MRCA of the perennial clade dates well to the middle of the Pleistocene (median = 1.09 mya; 95% HPD interval = 0.48–1.93 mya) [see SUPPORTING INFORMATION]. DISCUSSION Specific entities Genomic variation as evidenced by Bayesian mixture clustering of AFLP data (Fig. 2B) shows a basic concordance with morphological variation (S. Talavera et al., unpubl. data). Diagnostic characters for distinguishing H. comosa subsp. lusitanica from the nominal subspecies are provided by the involucral bracts of the heads. In the former, the subapical appendix of the inner involucral bracts is shorter (1–3.5 mm long in H. comosa subsp. lusitanica vs. 5–8 mm long in H. comosa subsp. comosa) and projects less above the bracts (1–2 mm in H. comosa subsp. lusitanica vs. 2–6 mm in H. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 20 comosa subsp. comosa). The outer involucral bracts usually have fewer pairs of marginal spines (2–4 in H. comosa subsp. lusitanica vs. 10–21 in H. comosa subsp. comosa). Similarly, diagnostic characters for distinguishing H. aculeata subsp. maroccana from the nominal subspecies are also provided by the involucral bracts of the heads. The outer involucral bracts of H. aculeata subsp. aculeata usually lack marginal spines. Only rarely do they have one or two pairs of marginal spines in addition to the terminal spine, whereas the outer involucral bracts of H. aculeata subsp. maroccana have considerably more pairs of marginal spines (10–21). Following De Queiroz (2007) in treating the existence as separately evolving segments of lineages as the only necessary property of species, we therefore propose to distinguish H. comosa subsp. lusitanica as a separate species from H. comosa and H. aculeata subsp. maroccana as a separate species from H. aculeata. As outlined above, lines of evidence for lineage separation come from AFLP data and morphology. The lineages also have distinct geographic distributions (Fig. 1) and ecological preferences (see below). In this sense, we will refer to H. comosa subsp. lusitanica as H. spinosa, comb. nov., and to H. aculeata subsp. maroccana as H. maroccana, comb. nov., in the following (the new combinations are presented in the section Taxonomic Implications). North-western African origin Although ancestral area reconstruction is not quite conclusive, it suggests that the perennial clade of Helminthotheca originated in western North Africa, possibly in the Middle Atlas or Rif. A caveat of the biogeographic analysis, however, is that few populations from central North Africa (Tell Atlas; at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 21 especially H. glomerata) and no populations from the High Atlas and Anti- Atlas have been included, so that we cannot more precisely determine the ancestral area. Our results for the perennial clade of Helminthotheca are well in line with observations in the closely related Hypochaeris sect. Hypochaeris (Ortiz et al. 2009). Both groups originated in western North Africa in the Pleistocene or Pliocene; their origins were thus associated with the ascent of the Mediterranean-type climate and/or Pleistocene climatic oscillations. The importance of the High and Middle Atlas region in addition to the Betic-Rif region for the evolution of Mediterranean plant taxa has been suggested by Médail and Quézel (1997). The High and Middle Atlas Mountains, just as the Rif, are known to have a high rate of endemism, which is explained by the long isolation and high altitude of these massifs, which made them serve as refugium for the Tertiary residual flora. In addition, there is also a large number of schizoendemics that demonstrate the role of the Atlas Mountains in more recent speciation processes (Médail and Quézel 1997). This is in line with climatic reconstructions, which suggest that a Mediterranean-type climate with summer drought was present in northern Africa earlier than in southern Europe (Quézel 1978; Suc 1984). Fauquette et al. (1999) describe Pliocene vegetation assemblages resembling the modern thermo-Mediterranean formation for the regions Andalusia (southern Spain), North Africa (Morocco, Algeria, Tunisia), and Sicily, corresponding to those regions inhabited by perennial species of Helminthotheca. In accordance with our age estimates, we might hypothesize that after the common ancestor of the perennial clade of Helminthotheca had originated in response to the ascent of the at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 22 Mediterranean-type climate in northern Africa, it held on during the Pliocene and diversified in the Pleistocene. Migratory routes out of western North Africa The evolutionary scenario derived from our data suggests two migratory routes out of western North Africa and across the Mediterranean Sea. The first expansion was to the north, across the Strait of Gibraltar, to the Iberian Peninsula. The second expansion was to the east, along central North African territories, to Sicily and the Apennine Peninsula. Numerous studies have investigated genetic differentiation between populations on both sides of the Strait of Gibraltar (e.g., Arroyo et al. 2008; Escudero et al. 2008), with differing outcomes. Whereas the Strait of Gibraltar has been an effective barrier to gene flow in Carex helodes after its dispersal from the Iberian Peninsula to North Africa (Escudero et al. 2008), extensive gene flow across the Strait of Gibraltar via seed dispersal has been documented in Calicotome villosa (Arroyo et al. 2008). In the central Mediterranean region, the Strait of Sicily separating North Africa and Sicily has also functioned as an important route of dispersal for plants and animals (e.g., Habel et al. 2008; Fernández-Mazuecos and Vargas 2011). Dispersal was probably facilitated by lowered sea water levels during the Pleistocene glacial periods (Thiede 1978). In Helminthotheca, seed dispersal could have been effectuated by wind or migratory birds (Finlayson 1992). Ecogeographic isolation As in other Mediterranean plant groups of Pliocene/Pleistocene age such as Antirrhinum (Vargas et al. 2009) and Reseda sect. Glaucoreseda (Martín-Bravo et al. 2010), there is also a clear geographic component to at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 23 speciation in the perennial clade of Helminthotheca. The biogeographic analysis suggests two lineage divergence events within western North Africa, the first yielding an Afro-Sicilian and an Afro-Iberian lineage and the second yielding H. maroccana (as progenitor) and H. aculeata (as derivative) within the Afro-Sicilian lineage. The placement of H. glomerata in this scenario still has to be determined. The third lineage divergence event is situated in the western Iberian region, yielding H. comosa (as progenitor) and H. spinosa (as derivative). Accurate identification of species pairs is crucial for investigating isolating barriers. The limit between the distributions of H. comosa and H. spinosa closely follows the Guadalquivir river basin, which separates the Mesozoic and Cenozoic Betic Cordillera ('Western Betic Region'; García- Barros et al. 2002) inhabited by H. comosa and the Paleozoic Iberian Massif ('Western Iberia') inhabited by H. spinosa. Characteristics of the two regions such as those associated with the substrate (calcareous in the Betic Cordillera, acidic in the Iberian Massif) are expected to exert divergent selective pressure on populations, resulting in ecogeographic isolation of the diverging lineages (Sobel et al. 2010). Ecogeographic isolation might also be hypothesized for the species pair H. aculeata/H. maroccana, in response to differences in substrate or precipitation. Helminthotheca aculeata grows on calcareous substrate of the Cenozoic, whereas H. maroccana grows on crystalline rocks and schist. Hybrids exist in contact zones between the regions. For example, some populations of H. comosa grow in calcareous enclaves, which exist in the western Iberian region. Hybrids between H. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 24 spinosa and H. comosa are found precisely in the ecotones between acidic and calcareous soils. As already supposed by Sauvage (1961), assessment of ploidy levels could shed more light on the issue of hybridization coupled with polyploidization in Helminthotheca. To the present day, there are few chromosome counts available, whereby a diploid chromosome number (2n = 10) has been reported for H. comosa and H. spinosa (Fernandes and Queirós 1971; Gallego 1981; Luque 1981). A H. comosa-like tetraploid population (2n = 20) has been found in northern Morocco (Talavera et al. 327/03, SEV237787; S. Talavera, unpubl. data), close to the here analysed hybrid population CM. Taxonomic implications As a result of the genetic analyses and the morphological survey of the populations included in this work (S. Talavera et al., unpubl. data), two new combinations and descriptions of three hybrids that are new to science are now necessary. New combinations: Helminthotheca maroccana (Sauvage) Talavera & Tremetsberger, comb. nov.: Picris aculeata subsp. maroccana Sauvage in Trav. Inst. Sci. Chérifien, Sér. Bot. 22: 202 (1961) ≡ Helminthotheca aculeata subsp. maroccana (Sauvage) Greuter in Willdenowia 33: 233 (2003). Ind. loc.: “Forêt des Bni- Âbid.” Holotype: herbarium C.S. [Charles Sauvage] no. 14689. Helminthotheca spinosa (DC.) Talavera & Tremetsberger, comb. nov.: Helminthia spinosa DC. in Candolle & Lamarck, Fl. Franç., ed. 3, 4: 58 (1805) ≡ Picris spinosa (DC.) Poir. in Lamarck, Encyc., Suppl. 4: 408 (1816). Ind. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 25 loc.: “Je décrit cette plante d’après des échantillons originaires des Pyrénées et qui proviennent de l’herbier de Lemonnier.” Lectotype, here designated: G00317181 [G-DC]. The type material that is conserved consists of the upper part of three plants, mounted on two “échantillons” or vouchers. One of the vouchers comprises two plants and the other one plant. In the voucher with two plants, there are also two handwritten labels. On one label, which possibly stems from the herbarium of Lemonnier, it is indicated “Picris aculeata Desf.?/P. sprengeriana Gaert.?/Pyrénées”, and on the other label, A. P. de Candolle indicated “Helminthia spinosa Fl. Fr.”. The other voucher, with a single plant, does not contain any label, but the plant is similar to the other two. We select as lectotype the plant mounted on the right side of the voucher that contains the two labels, the more complete of the two plants. The other plant of this voucher and the plant from the other voucher are isolectotypes. = Helminthotheca comosa subsp. lusitanica (Welw. ex Schltdl.) P. Silva & Escud. in Bol. Soc. Brot., sér. 2, 60: 156 (1987) [for synonyms see Greuter, Med-Checkl. 2: 240 (2008)]. New hybrids: Helminthotheca × hispanica Tremetsberger & Talavera, hybr. nov.: Helminthotheca comosa (Boiss.) Holub × H. spinosa (DC.) Talavera & Tremetsberger Holotype: SEV237782. Spain, Huelva, Hinojos, “Las Porqueras”, in Quercus suber forest, 80 m, 37.29ºN-6.42ºW, 30 June 2007, S. Talavera, nº 228/07. Isotypes: SEV237783, SEV237784. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 32 García-Barros E, Gurrea P, Luciáñez MJ, Cano JM, Munguira ML, Moreno JC, Sainz H, Sanz MJ, Simón JC. 2002. Parsimony analysis of endemicity and its application to animal and plant geographical distributions in the Ibero-Balearic region (western Mediterranean). Journal of Biogeography 29:109–124. Goertzen LR, Cannone JJ, Gutell RR, Jansen RK. 2003. ITS secondary structure derived from comparative analysis: implications for sequence alignment and phylogeny of the Asteraceae. Molecular Phylogenetics and Evolution 29:216–234. Habel JC, Meyer M, El Mousadik A, Schmitt T. 2008. Africa goes Europe: The complete phylogeography of the marbled white butterfly species complex Melanargia galathea/M. lachesis (Lepidoptera: Satyridae). Organisms, Diversity & Evolution 8:121–129. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95–98. Heled J, Drummond AJ. 2010. Bayesian inference of species trees from multilocus data. Molecular Biology and Evolution 27:570–580. Hijmans RJ, Guarino L, Cruz M, Rojas E. 2001. Computer tools for spatial analysis of plant genetic resources data: 1. DIVA-GIS. Plant Genetic Resources Newsletter 127:15–19. Hsü KJ, Ryan WBF, Cita MB. 1973. Late Miocene desiccation of the Mediterranean. Nature 242:240–244. Huson DH, Bryant D. 2006. Application of phylogenetic networks in evolutionary studies. Molecular Biology and Evolution 23:254–267. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 33 Lack HW. 1974. Die Gattung Picris L., sensu lato, im ostmediterranwestasiatischen Raum. Dissertationen der Universität Wien, Band 116. Wien: Verband der wissenschaftlichen Gesellschaften Österreichs (VWGÖ). Lack HW. 1975. A note on Helminthotheca Zinn (Compositae). Taxon 24:111– 112. Lonergan L, White N. 1997. Origin of the Betic-Rif mountain belt. Tectonics 16:504–522. Luque T. 1981. Números cromosómicos para la flora Española. Números 210-213. Lagascalia 10:236–237. Martín-Bravo S, Valcárcel V, Vargas P, Luceño M. 2010. Geographical speciation related to Pleistocene range shifts in the western Mediterranean mountains (Reseda sect. Glaucoreseda, Resedaceae). Taxon 59:466–482. Médail F, Quézel P. 1997. Hot-spots analysis for conservation of plant biodiversity in the Mediterranean basin. Annals of the Missouri Botanical Garden 84:112–127. Médail F, Diadema K. 2009. Glacial refugia influence plant diversity patterns in the Mediterranean Basin. Journal of Biogeography 36:1333–1345. Müller K. 2005. SeqState: primer design and sequence statistics for phylogenetic DNA datasets. Applied Bioinformatics 4:65–69. Nei M, Li WH. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences, USA 76:5269–5273. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 34 Ortiz MÁ, Tremetsberger K, Stuessy TF, Terrab A, García-Castaño JL, Talavera S. 2009. Phylogeographic patterns in Hypochaeris section Hypochaeris (Asteraceae, Lactuceae) of the western Mediterranean. Journal of Biogeography 36:1384–1397. Pignatti S. 1982. Flora d’Italia. Volume 3. Bologna: Edagricole. Quézel P, Santa S. 1963. Nouvelle Flore de l´Algérie et des régions désertiques méridionales. Paris: Centre National de la Recherche Scientifique. Quézel P. 1978. Analysis of the flora of Mediterranean and Saharan Africa. Annals of the Missouri Botanical Garden 65:479–534. Rambaut A, Drummond AJ. 2003-2005. Tracer, ver. 1.3, available from http://evolve.zoo.ox.ac.uk/beast/. Rambaut A. 2006-2009. FigTree, ver. 1.3.1, available from http://tree.bio.ed.ac.uk/software/figtree/. Ree RH, Smith SA. 2008. Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis. Systematic Biology 57:4–14. Sang T, Crawford DJ, Stuessy TF. 1997. Chloroplast DNA phylogeny, reticulate evolution and biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84:1120–1136. Sauvage C. 1961. Flore des subéraies marocaines. Catalogues des cryptogames vasculaires et des phanerogames. Travaux de l’Institut Scientifique Chérifien, Série Botanique 22:1–252. Schlüter PM, Harris SA. 2006. Analysis of multilocus fingerprinting data sets containing missing data. Molecular Ecology Notes 6:569–572. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 35 Schönswetter P, Tribsch A. 2005. Vicariance and dispersal in the alpine perennial Bupleurum stellatum L. (Apiaceae). Taxon 54:725–732. Shaw J, Lickey EB, Schilling EE, Small RL. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. American Journal of Botany 94:275–288. Sobel JM, Chen GF, Watt LR, Schemske DW. 2010. The biology of speciation. Evolution 64:295–315. Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML Web-Servers. Systematic Biology 75:758–771. Stoesser G, Baker W, Van den Broek A, Garcia-Pastor M, Kanz C, Kulikova T, Leinonen R, Lin Q, Lombard V, Lopez R, Mancuso R, Nardone F, Stoehr P, Tuli M, Tzouvara K, Vaughan R. 2003. The EMBL Nucleotide Sequence Database: major new developments. Nucleic Acids Research 31:17–22. Suc JP. 1984. Origin and evolution of the Mediterranean vegetation and climate in Europe. Nature 307:429–432. Swofford DL. 2003. PAUP*: Phylogenetic analyses using parsimony (*and other methods), ver. 4.0b10. Sunderland: Sinauer. Thiede J. 1978. A Glacial Mediterranean. Nature 276:680–683. Tremetsberger K, Urtubey E, Terrab A, Baeza CM, Ortiz MÁ, Talavera M, König C, Temsch EM, Kohl G, Talavera S, Stuessy TF. 2009. Pleistocene refugia and polytopic replacement of diploids by tetraploids in the Patagonian and Subantarctic plant Hypochaeris incana (Asteraceae, Cichorieae). Molecular Ecology 18:3668–3682. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 36 Tremetsberger K, Gemeinholzer B, Zetzsche H, Blackmore S, Kilian N, Talavera S. 2013. Divergence time estimation in Cichorieae (Asteraceae) using a fossil-calibrated relaxed molecular clock. Organisms, Diversity & Evolution 13:1–13. Vargas P, Carrió E, Guzmán B, Amat E, Güemes J. 2009. A geographical pattern of Antirrhinum (Scrophulariaceae) speciation since the Pliocene based on plastid and nuclear DNA polymorphisms. Journal of Biogeography 36:1297–1312. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 315–322. Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31:3406–3415. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 37 FIGURE LEGENDS Figure 1. Distribution ranges of H. aculeata subsp. aculeata, H. aculeata subsp. maroccana (H. maroccana, comb. nov.), H. comosa subsp. comosa, and H. comosa subsp. lusitanica (H. spinosa, comb. nov.) and populations analysed in this study (colour of triangles refers to belonging of accessions to one of four clusters inferred by Bayesian mixture clustering of AFLP data). Figure 2. Results of AFLP analysis showing division of population samples of perennial species of Helminthotheca into four clusters. A, Phylogenetic network (colour coding according to Bayesian mixture clustering). B, Bayesian admixture clustering with evidence for admixture in populations CM and CS. C, Neighbour-joining tree including the outgroup, H. echioides (colour coding according to Bayesian mixture clustering). Numbers above branches indicate bootstrap support values >50%. Figure 3. DensiTree of the perennial clade of Helminthotheca showing the complete tree set (thin lines) and consensus trees (thick lines; 10% burn-in) from (A) SNAPP analysis of AFLP data (single consensus tree) and (B) *BEAST analysis of ITS and plastid DNA sequences. The posterior probability of the H. aculeata subsp. maroccana/H. aculeata subsp. aculeataclade is 58%. With coded indels of plastid DNA sequences included, it increases to 92% (not shown). Figure 4. Phylogenetic trees of perennial species of Helminthotheca, with H. echioides designated as outgroup. A, Best-scoring ML tree of ITS1, 5.8S rDNA, and ITS2 sequences. B, Bootstrap 50% majority rule consensus tree (plus other groups compatible with it) based on MP analysis of concatenated at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 38 ndhF-rpl32 and rpl32-trnL sequences. Numbers above branches indicate bootstrap support values >70%. Colour coding refers to belonging of individuals to one of four groups obtained by Bayesian mixture clustering of AFLP data. Figure 5. Model-based ancestral area reconstruction in the perennial clade of Helminthotheca using the software Lagrange (Ree and Smith 2008). Global - lnL at root node is 9.405, the estimated dispersal rate is 1.124, and the estimated local extinction rate is 4.285e-09. A, Area adjacency matrix used for input. B, Species tree (consensus tree of *BEAST analysis) displaying most probable ancestral areas. C, Splits with associated log likelihoods (lnL) and relative probabilities (Rel. Prob.) for each node. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 39 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 40 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from 41 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from Silves and Sao Marcos da Serra (37.30° N, 8.40° W), Talavera et al. 224/06 LN830763 identical individuals), LN830860 identical individuals), LN830818 Pop. S6 Portugal, Beja District (W Iberia): Serra de Monchique between Monchique and Odemira (37.39° N, 8.49° W), Talavera et al. 244/06 LN830764, LN830765 LN830861 (two identical individuals), LN830862 LN830819 (three identical individuals) Pop. S7 Portugal, Setubal District (W Iberia): Serra da Arrábida between Setubal and Portinho da Arrábida (38.47° N, 9.00° W), Talavera et al. 262/06 LN830766, LN830767 LN830863 (three identical individuals) LN830820 (three identical individuals) Pop. S8 Portugal, Coimbra District (W Iberia): Figueira da Foz, Cabo Mondego (40.18° N, 8.90° W), Talavera et al. 263/06 LN830768, LN830769 LN830864 (two identical individuals), LN830865 LN830821 (two identical individuals), LN830822 Helminthotheca echioides (L.) Holub Pop. E1 Portugal, Coimbra District: Figueira da Foz, Cabo Mondego (40.18° N, 8.90° W), Talavera et al. 266/06 LN830755 LN830854 LN830812 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from Pop. E2 Spain, Huelva Province: between Gibraleón and Cartaya (37.33° N, 7.03° W), Tremetsberger & Ortiz 2/06 LN830756 LN830855 LN830813 Pop. E3 Spain, Cádiz Province: El Palmar (36.32° N, 6.08° W), Talavera & Ortiz s/n (26 April 2006) LN830757 LN830856 LN830814 Pop. E4 Morocco, Tangier-Tetouan Region: S of Ksar es-Seghir (35.77° N, 5.53° W), Talavera et al. 23/07 LN830758 (four identical individuals) – – Pop. E5 Algeria, Annaba Province: El Kerma on road Annaba-Guelma (36.70° N, 7.62° E), Véla s/n (5 June 2006) LN830759 LN830857 LN830815 Helminthotheca × hispanica Tremetsberger & Talavera, hybr. nov. Pop. CS Spain, Huelva Province (W Iberia): Hinojos (37.29° N, 6.42° W), Talavera 228/07 LN830772, LN830773 LN830868, LN830869 (two identical individuals) LN830824 (three identical individuals) Helminthotheca × riphaea Tremetsberger & Talavera, hybr. nov. at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from Pop. AM1 Morocco, Taza-Al Hoceima-Taounate Region (Rif): between Targuist and Al Hoceima (35.02° N, 4.17° W), Rico et al. MS-1099 LN830801, LN830802 LN830888 (three identical individuals) LN830844 (three identical individuals) Pop. AM2 Morocco, Taza-Al Hoceima-Taounate Region (Rif): between Tafersit and Temsaman (35.04° N, 3.59° W), Rico et al. MS-1145 LN830803, LN830804 LN830889 (three identical individuals) LN830845 (three identical individuals) Helminthotheca × tingitana Tremetsberger & Talavera, hybr. nov. Pop. CM Morocco, Tangier-Tetouan Region (Rif): between Chefchaouen and Tetouan (35.37° N, 5.38° W), Rico et al. MS-999 LN830793, LN830794 LN830880 (two identical individuals), LN830881 LN830836 (three identical individuals) at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from Table 2. Estimates of divergence and diversity derived from AFLP data in populations of Helminthotheca. N, sample size; ND, not determined Species and region N Estimates of divergence Within-population genetic diversity Population Nprivate bands Rarity index H. aculeata (Vahl) Lack subsp. aculeata, Algeria and Sicily A3 2 2 3.5 ND A4 2 2 3.0 ND A5 5 6 3.3 0.099 Mean ± SD 3.3 ± 2.3 3.3 ± 0.3 0.099 H. × riphaea Tremetsberger & Talavera, hybr. nov., Morocco AM1 5 3 2.9 0.098 AM2 2 9 7.4 ND Mean ± SD 6.0 ± 4.2 5.2 ± 3.2 0.098 H. aculeata subsp. maroccana (Sauvage) Greuter [H. maroccana (Sauvage) Talavera & Tremetsberger, comb. nov.], Morocco M1 5 1 2.3 0.113 M2 3 4 3.3 0.115 M3 5 4 4.3 0.121 Mean ± SD 3.0 ± 1.7 3.3 ± 1.0 0.116 ± 0.004 H. × tingitana Tremetsberger & Talavera, hybr. nov., Morocco CM 5 1 2.5 0.109 H. comosa (Boiss.) Holub subsp. comosa, Spain C3 3 7 4.5 0.069 C4 5 3 2.3 0.068 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from C5 5 0 1.4 0.104 C6 5 3 2.1 0.097 Mean ± SD 3.3 ± 2.9 2.6 ± 1.3 0.084 ± 0.019 H. × hispanica Tremetsberger & Talavera, hybr. nov., Spain CS 5 ND ND 0.100 H. comosa subsp. lusitanica (Welw. ex Schltdl.) P. Silva & Escud. [H. spinosa (DC.) Talavera & Tremetsberger, comb. nov.], Spain and Portugal S1 5 ND ND 0.103 S2 5 1 1.9 0.096 S3 5 ND ND 0.080 S4 5 ND ND 0.086 S5 5 5 2.7 0.097 S7 5 0 1.7 0.055 S8 5 3 2.2 0.083 Mean ± SD 2.3 ± 2.2 2.1 ± 0.5 0.087 ± 0.015 at UNIVERSIDAD DE SEVILLA on November 15, 2016http://aobpla.oxfordjournals.org/Downloaded from