Journal of Biogeography (J. Biogeogr.) (2009) 36, 1297–1312 SPECIAL A geographical pattern of Antirrhinum ISSUE (Scrophulariaceae) speciation since the Pliocene based on plastid and nuclear DNA polymorphisms Pablo Vargas1*, Elena Carrio´ 2, Beatriz Guzma´n1, Elena Amat1 and Jaime Gu¨emes2 1Real Jardı´n Bota´nico de Madrid, CSIC, ABSTRACT Madrid, Spain and 2Jardı´ Botanic de Valencia, Aim To infer phylogenetic relationships among Antirrhinum species and to calle Quart 80, Universitat de Valencia, Valencia, Spain reconstruct the historical distribution of observed sequence polymorphism through estimates of haplotype clades and lineage divergence. Location Antirrhinum is distributed primarily throughout the western Mediterranean, with 22 of 25 species in the Iberian Peninsula. Methods Plastid (83 trnS-trnG and 83 trnK-matK) and nuclear (87 ITS) sequences were obtained from 96 individuals representing 24 of the 25 Antirrhinum species. Sequences were analysed using maximum parsimony, Bayesian inference and statistical parsimony networking. Molecular clock estimates were obtained for plastid trnK-matK sequences using the penalized likelihood approach. Results Phylogenetic results gave limited support for monophyletic groups within Antirrhinum. Fifty-one plastid haplotypes were detected and 27 missing haplotypes inferred, which were all connected in a single, star-like network. A significant number of species shared both the same haplotypes and the same geographical areas, primarily in eastern Iberia. Furthermore, many species harboured populations with unrelated haplotypes from divergent haplotype clades. Plastid haplotype distribution, together with nucleotide additivity in 59 of the 86 nuclear ribosomal ITS sequences, is interpreted as evidence of extensive hybridization. Lineage divergence estimates indicated that differentiation within Antirrhinum post-dates the Miocene, when the Mediterranean climate was established. Main conclusions Incongruence between plastid sequences, nuclear sequences and taxonomic delimitation is interpreted as strong evidence of limited cladogenetic processes in Antirrhinum. Rather, extensive nucleotide additivities in ITS sequences in conjunction with haplotype and haplotype-clade distributions related to geographical areas support both recent and ancient hybridization. This geographical pattern of Antirrhinum speciation, particularly in eastern Iberia, is *Correspondence: Pablo Vargas, Real Jardı´n congruent with isolation–contact–isolation processes in the Pleistocene. ´ Botanico de Madrid, Consejo Superior de Keywords Investigaciones Cientı´ficas (CSIC), Plaza de Murillo 2, 28014 Madrid, Spain. Hybridization, Iberian Peninsula, ITS, Mediterranean, phylogeny, phylogeogra- E-mail: [email protected] phy, trnK-matK, trnS-trnG. Mediterranean (Fig. 1). The circumscription of Antirrhinum INTRODUCTION into these species represents the result of more than 250 years The genus Antirrhinum L. (snapdragons) contains approxi- of taxonomic effort. Linnaeus (1753) considered 28 species in mately 25 species primarily distributed throughout the western Antirrhinum, of which only A. majus and A. molle are currently ª 2009 The Authors www.blackwellpublishing.com/jbi 1297 Journal compilation ª 2009 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2008.02059.x P. Vargas et al. (a) (b) Figure 1 Geographical distribution of the 25 Antirrhinum species. (a) Species with broader distributions (A. controversum, A. cirrhige- rum, A. latifolium, A. litigiosum, A. majus, A. meonanthum, A. siculum, A. tortuosum) and A. mollissimum. (b) Species primarily distributed in the Iberian Peninsula (A. australe, A. braun-blanquetii, A. charidemi, A. graniticum, A. grosii, A. hispanicum, A. linkianum, A. lopesianum, A. microphyllum, A. molle, A. pertegasii, A. pulverulentum, A. sempervirens, A. subbaeticum, A. valentinum) and A. latifolium. All species were sampled, except for the narrowly distributed endemic A. martenii (northern Africa). Dashed lines divide the Iberian Peninsula into the four quadrants used in the phylogeographical analysis. retained in this genus. Increasing numbers of species were different numbers of species recognized by more recent proposed in further publications by Willdenow (1800; four authors: 23 in Rothmaler (1956); 24 in Stubbe (1966); 17 in species) and Bentham (1846; eight species). The complex Webb (1972); and 20 in Sutton (1988). This illustrates an phenotype delimitation is reflected in the unstable classifica- ongoing discussion of taxonomic entities, which are charac- tion of certain populations into different taxa and in the terized by a combination of few morphological characters (11) 1298 Journal of Biogeography 36, 1297–1312 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd Geographical speciation in Antirrhinum shared by two or more species (Vargas et al., 2004). Based on MATERIALS AND METHODS the distribution of key morphological characters, such as plant indumentum, leaf and bract shape, and branching patterns, Sampling strategy and DNA sequencing Rothmaler (1956) and Webb (1972) envisioned isolation– contact–isolation processes during the dry and wet episodes of A total of 96 individuals representing 24 of the 25 Antirrhinum the Ice Ages, resulting in hybridization and subsequent species were sampled (Table 1). The number of populations character sharing. sampled per species depended on their distribution and Despite the lack of taxonomic consensus and the fact that intraspecific variation. Particular effort was made to associate species boundaries are difficult to define, researchers have species distribution, morphological forms and taxonomic not been impeded in investigating evolution in Antirrhinum. nomenclature by analysing material from 10 localities (locus The inheritance of floral development in A. majus was classicus) where plants were first collected for the original studied by Darwin (1876) and Mendel (1865), and the species descriptions. Based on previous phylogenetic results, pioneering work of Bateson, Wheldale and particularly Baur the tribe Antirrhineae (i.e. 29 genera including Antirrhinum)is established Antirrhinum as a model species group from the monophyletic (Vargas et al., 2004) and closely related to the beginning of twentieth century onwards (Schwarz-Sommer Plantaginaceae (Olmstead et al., 2001). In agreement with et al., 2003). Baur was also one of the first researchers to these results, outgroup sequences of genera closely related appreciate the potential of evolutionary genetics, and used to Antirrhinum (Acanthorrhinum, Pseudomisopates, Misopates, Antirrhinum species and mutant lines to identify and map Gambelia, Chaenorhinum) and sequences of Plantago and genes responsible for differences in flower colour and Digitalis were generated and analysed (Table 1). morphology (Stubbe, 1966). The combination of classical Total DNA was extracted from silica-dried material using genetics using inbred lines and new genetic technologies the CTAB (cetyl trimethyl ammonium bromide) method as (Harrison & Carpenter, 1979) has resulted in a concerted in Vargas et al. (2004) or DNeasy Plant Mini Kits (Qiagen, collaborative effort between several research groups to Valencia, CA, USA). Polymerase chain reactions (PCRs) were describe a model system of floral morphogenesis (Coen performed on a Perkin-Elmer PCR System 9700 (Fremont, et al., 1986; Endress, 1992; Schwarz-Sommer et al., 2003; CA, USA) or a MJ Research (Waltham, MA, USA) thermal Whibley et al., 2006). cycler. We obtained and analysed 83 trnK-matK,83trnS-trnG Research effort in Antirrhinum also includes intraspecific, and 87 ITS sequences (Table 1). Standard primers were used population genetic studies, with 12 out of 25 species for amplification of the trnK-matK spacer (trnK-3914F, examined by means of a range of molecular markers matK-1470R) (Johnson & Soltis, 1994) and the trnS (Mateu-Andre´s, 1999; Mateu-Andre´s & Segarra-Moragues, (GCU)-trnG (UCC) spacer (Hamilton, 1999). After 1–3 min 2000; Torres et al., 2003; Mateu-Andre´s & de Paco, 2006). denaturation at 94°C, PCR conditions were: 28–30 cycles of Considerable population diversity has been uncovered, as 1 min at 94°C, and 1–2 min at 50–58°C, followed by an evidenced by a wide range of gene heterozygosity values extension of 10 min at 72°C. One microlitre of dimethyl (HT: 0.03–0.52) from co-dominant allozymes (Mateu-Andre´s, sulfoxide (DMSO) at 99.9 % was included in each 25-lL 1999; Mateu-Andre´s & de Paco, 2006), and similar high levels reaction. Amplified products were cleaned using spin filter of diversity revealed by dominant, fingerprinting techniques columns (PCR Clean-up Kit, MoBio Laboratories, Carlsbad, (Jime´nez et al., 2005a). This diversity has been shown to be CA, USA) following the manufacturer’s protocols. Cleaned both congruent (Jime´nez et al., 2005a) and incongruent products were then directly sequenced using dye terminators (Vargas et al., 2004; Jime´nez et al., 2005b) with currently (Big Dye Terminator ver. 2.0, Applied Biosystems, Little recognized taxonomic species. Chalfont, UK) following the manufacturer’s protocols and The body of knowledge on the taxonomy, population run into polyacrylamide electrophoresis gels (7%) using an genetics and genetic control of organ morphogenesis in Applied Biosystems Prism Model 3700 automated sequencer. Antirrhinum is in contrast to the limited information available PCR primers were used for cycle sequencing. Sequence data regarding phylogenetic relationships and phylogeographical were assembled and