A Geographical Pattern of Antirrhinum

Home , Antirrhineae, Antirrhinum, Antirrhinum majus, Callitriche, Chaenorhinum, Misopates

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 Jardıń Botańico 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 Jardıń 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. cirrhigerum, 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 94C, PCR conditions were: 28–30 cycles of Considerable population diversity has been uncovered, as 1 min at 94C, and 1–2 min at 50–58C, followed by an evidenced by a wide range of gene heterozygosity values extension of 10 min at 72C. 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, (Jimeńez et al., 2005a). This diversity has been shown to be CA, USA) following the manufacturer’s protocols. Cleaned both congruent (Jimeńez et al., 2005a) and incongruent products were then directly sequenced using dye terminators (Vargas et al., 2004; Jimeńez 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 edited using the program seqed patterns. Limited resolution obtained in previous phylogenetic (Applied Biosystems, Foster City, CA, USA). Procedures used studies of Antirrhinum species based on nuclear ribosomal ITS for DNA sequencing of the ITS region are given in Vargas (Vargas et al., 2004) and plastid (Jimeńez et al., 2005b) DNA et al. (2004). IUPAC (International Union of Pure and sequences leaves open the question of whether complex and Applied Chemistry) symbols were used to represent nucleo- recent evolutionary processes or unsuitable sampling and tide ambiguities. molecular markers are responsible for the poor patterns observed. Here, we performed phylogenetic, phylogeographical Plastid haplotype analysis and lineage divergence dating analyses on an extended sample of Antirrhinum species using nuclear (ITS) and plastid (trnS- The 83 concatenated sequences of the trnK-matK and trnS- trnG, trnK-matK) sequences to address speciation and diver- trnG spacers were aligned by hand, given the low number of gence times in a morphologically complex Mediterranean indels across sequences. The number of Antirrhinum plastid system. haplotypes and relationships among them were inferred using

Journal of Biogeography 36, 1297–1312 1299 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd 1300 Table 1 Antirrhinum material used for ITS, trnS-trnG and trnK-matK sequencing of 96 samples. Population numbers are given in brackets after species names. Geographical abbreviations: Vargas P. SE, south-eastern Iberia; SW, south-western Iberia; NE, north-eastern Iberia; NW, north-western Iberia; NA, northern Africa; E, Europe (excluding Iberia) and western Asia. Localities in bold indicate sites where the material was collected for the ﬁrst plant description (locus classicus). Voucher abbreviations: BdB, Valencia database; ECA, Elena Carrio´’s collection numbers;

JG, Jaime Gu¨emes’ collection numbers; PV, Pablo Vargas’ collection numbers; JRV, Jesu´ s Riera’s collection numbers; MA, herbarium of the Royal Botanic Garden of Madrid; MS, Marı´a Santos’ al. et collection numbers; GB, Gianluigi Bacchetta’s collection numbers; LM, Leopoldo Medina’s collection numbers; VAL, herbarium of the Botanical Garden of Valencia. Collection vouchers in bold indicate plant material used in Vargas et al. (2004), from which nomenclature is followed except for A. controversum (= A. barrelieri). Asterisks (*) after GenBank accession numbers of ITS sequences refer to those from Vargas et al. (2004).

Number Collection GenBank accession no. of ITS Plastid Taxon Geographical area/locality voucher (ITS/trnS-trnG/trnK-matK) additivities haplotype no.

A. australe Rothm. (1) SE/Spain: Albacete, Yeste JG4077 EU677194 EU673477 EU717968 0 42 A. australe Rothm. (2) SW/Spain: Ca´diz, Benaocaz ECA49 EU677195 EU673478 EU717969 0 17 A. australe Rothm. (3) SE/Spain: Granada, Castril VAL 140895 AY731273* EU673479 EU717970 4 43 A. braun-blanquetii Rothm. (1) NW/Spain: Asturias, Cuetu L¢Abeyera JRV5333 EU677196 EU673480 EU717971 6 1 A. braun-blanquetii Rothm. (2) NW/Spain: Leoń, Oblanca JRV5177 EU677197 EU673481 EU717972 0 7 A. braun-blanquetii Rothm (3) NW/Spain: Palencia, Cervera de Pisuerga VAL35121 AY731269* – 0 – A. charidemi Lange (1) SE/Spain: Almerıá, C. Gata, La Lobera 132PV05(1) EU677198 EU673482 EU717973 0 37 A. charidemi Lange (2) SE/Spain: Almerıá, C. Gata, Sabinal 24PV05(16) – EU673483 EU717974 – 37 A. charidemi Lange (3) SE/Spain: Almerıá, C. Gata, Santa Cruz 136PV05(2) EU677199 EU673484 EU717975 0 37 ª

09TeAtos ora compilation Journal Authors. The 2009 A. charidemi Lange (4) SE/Spain: Almerıá, C. Gata, Vela Blanca 137PV05(7) – EU673485 EU717976 – 37 A. charidemi Lange (5) SE/Spain: Almerıá, C. Gata, Sabinal 24PV05 – EU673486 EU717977 – 37 A. charidemi Lange (6) SE/Spain: Almerıá, C. Gata, Santa Cruz 136PV05 (7) – EU673487 EU717978 – 37 A. charidemi Lange (7) SE/Spain: Almerıá, C. Gata, Vela Blanca 137PV05(5) FJ487611 EU673488 EU717979 0 49 A. charidemi Lange (8) SE/Spain: Almerıá, C. Gata VAL37158 AY731282* – 0 – A. cirrhigerum Welw. ex Ficalho (1) NA/Morocco: Doukkala-Abda, El Jadida VAL111299 EU677200 EU673489 EU717980 0 15 A. controversum Pau (1) SE/Spain: Albacete, Villa de Ves VAL 145152 AY731272* – 14 – A. controversum Pau (2) SE/Spain: Alicante, Jaloń BdB 47 EU677201 EU673491 EU717981 3 34 A. controversum Pau (3) SE/Spain: Almerıá, Berja ECA37 EU677202 EU673492 EU717982 4 40 A. controversum Pau (4) SE/Spain: Granada, Be´rchules BdB 15b EU677203 – 6 –

ora fBiogeography of Journal A. controversum Pau (5) SE/Spain: Valencia, Bolomor JG4001 EU677204 EU673494 EU717983 2 36 A. controversum Pau (6) SE/Spain: Valencia, Carcagente BdB 29 EU677205 EU673495 EU717984 8 34 A. controversum Pau (7) SE/Spain: Valencia, Chella JG4067 EU677206 EU673496 EU717985 3 35 ª A. controversum Pau (8) SE/Spain: Valencia, Xeresa, Colom BdB 2 EU677207 EU673497 EU717986 2 35 09BakelPbihn Ltd Publishing Blackwell 2009 A. graniticum Rothm. (1) SE/Spain: Madrid, Fuentiduen˜a del Tajo JG4009 AY731283* EU673498 EU717987 7 1 A. graniticum Rothm. (2) NE/Spain: Soria, Caltojar JG4101 EU677208 EU673499 EU717988 3 1 A. graniticum Rothm. (3) SW/Spain: Huelva, Aracena ECA54 EU677209 EU673500 EU717989 0 29 A. grosii Font Quer (1) NW/Spain: A´ vila, El Trampal ECA77/VAL37049 AY731281* EU673501 EU717990 2 31

36 A. grosii Font Quer (2) NW/Spain: A´ vila, Guisando 276PV06 EU677210 EU673502 EU717991 0 30 1297–1312 , A. hispanicum Chav. (1) SE/Spain: Granada, Juviles BdB 14 FJ487614 EU673503 EU717992 5 39 A. hispanicum Chav. (2) SE/Spain: Granada, Veleta road 120PV99 AY731286* EU673504 EU717993 0 38 A. hispanicum Chav. (3) SE/Spain: Granada, Ve´lez de Benaudalla ECA40 EU677211 EU673505 EU717994 0 41 ª Biogeography of Journal Table 1 Continued 09TeAtos ora compilation Journal Authors. The 2009

Number Collection GenBank accession no. of ITS Plastid Taxon Geographical area/locality voucher (ITS/trnS-trnG/trnK-matK) additivities haplotype no.

A. latifolium Mill. (1) NE/Spain: Girona, Collada de Toses JG4142 EU677212 EU673506 EU717995 2 11 A. latifolium Mill. (2) NE/Spain: Le´rida, Bapa` VAL144658 AY731274* EU673507 EU717996 5 11

36 A. latifolium Mill. (3) NE/Spain: Le´rida, Martinet JG4139 – EU673508 EU717997 – 11 1297–1312 , A. latifolium Mill. (4) E/Italy: Piamonte, Cuneo MS781 EU677213 – 1 – A. linkianum Boiss. (1) NW/Spain: La Corun˜a, Cedeira S. Ortiz (s.n.) EU677214 EU673510 EU717998 1 1 A. linkianum Boiss. (2) SW/Portugal: Peniche, Cabo Carvoeiro ALQ3435 EU677215 EU673511 EU717999 1 8 A. linkianum Boiss. (3) SW/Portugal: Trafaria, Almada ALQ4877 – EU673512 EU718000 – 8 A. linkianum Boiss. (4) SW/Portugal: Cintra VAL144655 AY731278* – 0 – ª

09BakelPbihn Ltd Publishing Blackwell 2009 A. litigiosum Pau (1) NE/Spain: Teruel, Griegos ECA74 EU677216 EU673513 EU718001 6 1 A. litigiosum Pau (2) SE/Spain: Valencia, Serra VAL144656 AY731271* EU673514 EU718002 7 5 A. litigiosum Pau (3) SE/Spain: Zaragoza, Nue´valos VAL31598 AY731277* – 0 – A. lopesianum Rothm. (1) NW/Portugal: Bragança, Alfaiaõ F. Amich & S. Bernardo (s.n.) – EU673515 EU718003 – 6 A. lopesianum Rothm. (2) NW/Portugal: Vimioso, Carçao F. Amich & S. Bernardo (s.n.) EU677217 EU673516 EU718004 3 6 A. lopesianum Rothm. (3) NW/Spain: Salamanca, Corporario F. Amich & S. Bernardo (s.n.) EU677218 EU673517 EU718005 2 1 A. majus L. (1) NE/Spain: Barcelona, Greìxer JG4150 FJ487615 EU673518 EU718006 10 11 A. majus L. (2) NE/Spain: Huesca, Panticosa JG4108 FJ648325 EU673519 EU718007 8 9 A. majus L. (3) NE/Spain: Le´rida, Valle d¢Arań VAL144657 AY731280* EU673520 EU718008 8 12 A. majus L. (4) NE/France: He´rault, St. Chinian 230PV06 FJ487616 EU673521 EU718009 0 10 A. majus L. (5) NE/France: Pyreńee´s Orientales, Salses VAL 39727 FJ487613 EU673522 EU718010 10 12 A. majus L. (6) NE/Spain: Gerona, La Molina 273PV06 EU677219 – 7 – A. meonanthum Hoffmans. & Link (1) NW/Spain: A´ vila, S. Gredos, El Tremedal 149PV99 AY731284* EU673530 EU718011 8 30 A. meonanthum Hoffmans. & Link (2) NE/Spain: Soria, Rıó Lobos JG4098 EU677220 EU673531 EU718012 2 29 A. microphyllum Rothm. (1) NE/Spain: Cuenca, Buendıá JG4024 EU677221 EU673532 EU718013 0 24 A. microphyllum Rothm. (2) NE/Spain: Guadalajara, Bolarque JG4021 EU677222 EU673533 EU718014 3 24

A. microphyllum Rothm. (3) NE/Spain: Guadalajara, Sacedoń JG4023 EU677223 EU673534 EU718015 7 26 in speciation Geographical A. microphyllum Rothm. (4) NE/Spain: Guadalajara, Entrepenãs VAL40051 AY731267* – 2 – A. molle L. (1) NE/Spain: Barcelona, Rigureìxer JG4143 FJ487612 EU673524 EU718016 6 11 A. molle L. (2) NE/Spain: Huesca, Sopeira VAL35176 AY731268* EU673525 EU718017 1 12 A. mollissimum Rothm. (1) SE/Spain: Almerıá, Benizaloń ECA29 EU677224 EU673526 EU718018 4 37 A. mollissimum Rothm. (2) SE/Spain: Almerıá, Caballar gorge ECA32 EU677225 EU673527 EU718019 8 37 A. mollissimum Rothm. (3) SE/Spain: Almerıá, Sierra de Ga´dor VAL37143 AY731275* – 0 – A. pertegasii Rotmh. (1) NE/Spain: Castelloń, Cova Fosca JG4092 EU677226 EU673528 EU718020 0 4 A. pertegasii Rotmh. (2) NE/Spain: Castelloń, Sola` d’en Brull JG4091 EU677227 EU673529 EU718021 8 4 A. pertegasii Rotmh. (3) NE/Spain: Castelloń JG EU677228 – 0 – Antirrhinum A. pulverulentum La´zaro Ibiza (1) NE/Spain: Cuenca, Hoz de Beteta ECA28 EU677229 EU673535 EU718022 0 27 A. pulverulentum La´zaro Ibiza (2) NE/Spain: Guadalajara, Duroń JG4027 EU677230 EU673536 EU718023 0 3

1301 A. pulverulentum La´zaro Ibiza (3) NE/Spain: Guadalajara, La Peregrina JG4035 EU677231 EU673537 EU718024 3 51 1302 Table 1 Continued Vargas P.

Number Collection GenBank accession no. of ITS Plastid tal. et Taxon Geographical area/locality voucher (ITS/trnS-trnG/trnK-matK) additivities haplotype no.

A. pulverulentum La´zaro Ibiza (4) NE/Spain: Guadalajara, Peralejos Truchas ECA26 EU677232 EU673538 EU718025 4 25 A. pulverulentum La´zaro Ibiza (5) NE/Spain: Teruel, Tramacastilla ECA71 EU677233 EU673539 EU718026 1 28 A. pulverulentum La´zaro Ibiza (6) NE/Spain: Guadalajara, Alcorlo JG4028 EU677234 EU673540 EU718027 5 50 A. pulverulentum La´zaro Ibiza (7) NE/Spain: Zaragoza, Nue´valos VAL31592 AY731279* EU673541 EU718028 1 28 A. sempervirens Lapeyr. (1) NE/Spain: Huesca, Bielsa JG4114 EU677235 EU673542 EU718029 4 13 A. sempervirens Lapeyr. (2) NE/Spain: Huesca, Plan JG4116 EU677236 EU673543 EU718030 0 2 A. sempervirens Lapeyr. (3) NE/Spain: Huesca, Panticosa VAL145148 AY731270* EU673544 EU718031 0 14 A. siculum Mill. (1) E/Italy: Sicily, Catania GB66/06 EU677237 EU673545 EU718032 3 23 A. siculum Mill. (2) E/Italy: Sicily, Messine VAL 119899 / JG3019 AY731276* EU673546 EU718033 1 22 A. siculum Mill. (3) E/Italy: Sicily, Siracusa VAL 178308 / JG3437 FJ648327 EU673547 EU718034 1 21 A. siculum Mill. (5) NA/Morocco: Oriental, Zegzel 192PV00 EU677238 – 3 – A. subbaeticum Guëmes, Mateu & Sańchez Go´mez (1) SE/Spain: Albacete, Bogarra, El Batań JG4081 AY731287* EU673549 EU718035 0 25 A. subbaeticum Guëmes, Mateu & Sańchez Go´mez (2) SE/Spain: Albacete, Los Vizcaıńos JG4084 EU677239 EU673550 EU718036 0 25 A. subbaeticum Guëmes, Mateu & Sańchez Go´mez (3) SE/Spain: Murcia, Benı´zar JG4068 EU677240 EU673551 EU718037 1 33 A. subbaeticum Guëmes, Mateu & Sańchez Go´mez (4) SE/Spain: Murcia, Hondares BdB 227 EU677241 EU673552 EU718038 1 32 ª

09TeAtos ora compilation Journal Authors. The 2009 A. tortuosum Bosc ex Vent. (1) E/Italy: Sardinia, Cagliari GB136/06 FJ487617 EU673553 EU718039 10 16 A. tortuosum Bosc ex Vent. (2) NA/Morocco: West Rif, Talembot ALQ3441 EU677242 EU673554 EU718040 16 18 A. tortuosum Bosc ex Vent. (3) E/Turkey: Sulcuk, Efeso GB316/06 FJ487618 EU673555 EU718041 5 16 A. tortuosum Bosc ex Vent. (4) NA/Morocco: Taza-Al Hoceima, Taza 188PV06 FJ487619 EU673556 EU718042 2 18 A. tortuosum Bosc ex Vent. (6) E/Italy: Ancona, Sirolo VAL 39871 AY731285* – 1 – A. tortuosum Bosc ex Vent. (7) NA/Morocco: Taza-Al Hoceima, Tazzeka MA 643294/ 201PV06 – EU673558 EU718043 – 20 A. tortuosum Bosc ex Vent. (8) NA/Morocco: Tadla-Azilal, Ighir MA 746269/ LM3678 – EU673559 EU718044 – 19 A. tortuosum Bosc ex Vent. (9) E/Turkey: Bursa Ili, Gemlik 164PV06 FJ648326 EU673560 EU718045 5 16 A. valentinum Font Quer (1) SE/Spain: Valencia, Bolomor BdB 229 EU677243 EU673561 EU718046 0 47 A. valentinum Font Quer (2) SE/Spain: Valencia, Buixcarro´ JG4002 EU677244 EU673562 EU718047 0 44

ora fBiogeography of Journal A. valentinum Font Quer (3) SE/Spain: Valencia, Font del Cirer BdB 8 EU677245 EU673563 EU718048 5 45 A. valentinum Font Quer (4) SE/Spain: Valencia, La Drova JG4004 EU677246 EU673564 EU718049 2 48 A. valentinum Font Quer (5) SE/Spain: Valencia, Pen˜a Colom BdB 1 AY39799* EU673565 EU718050 1 46 ª Acanthorrhinum ramosissimum Rothm. Morocco: Mid Atlas JG 3284-2 – – EU718051 – – 09BakelPbihn Ltd Publishing Blackwell 2009 Chaenorhinum crassifolium (Cav.) Lange Spain: Huesca, Sopeira JG – – EU718052 – – Digitalis thapsi L. Spain: Madrid, Colmenar Viejo 61PV07 – – EU718053 – – Gambelia speciosa Nutt. Botanischer Garten Berlin-Dahlen VAL 145156 – – EU718054 – – Misopates orontium (L.) Raf. Spain: Valencia, Serra VAL 145155 – – EU718055 – –

36 Plantago lanceolata L. Spain: Madrid, Colmenar Viejo 62PV07 – – EU718056 – – 1297–1312 , Pseudomisopates rivas-martinezii (Sańchez Mata) Guëmes Spain: A´ vila, Sierra de Gredos 377PV99 – – EU718057 – – Geographical speciation in Antirrhinum the software tcs ver. 1.21 (Clement et al., 2000). The program To check the constancy of substitution rates, we used the implements a statistical parsimony approach using the Langley and Fitch (LF) test (Magalloń & Sanderson, 2005). As algorithm described in Templeton et al. (1992) to construct the null hypothesis of constant rate was rejected, we used the haplotype networks. The maximum number of differences penalized likelihood method (PL, Sanderson, 2002) imple- among haplotypes, as a result of single substitutions, was mented in r8s ver. 1.71. PL was implemented with the calculated with 95% confidence limits, and treating gaps as truncated Newton (TN) algorithm. Initial results were missing data. Given the distribution of Antirrhinum species obtained under the following parameters: cvstart = 0.5; (Fig. 1), we used six geographical areas (recognized biogeo- cvinc = 0.5; cvnum = 10 with cross-validation enforced. The graphical areas) to link haplotypes and geography: four Iberian rate smoothing with the lowest cross-validation scores was quadrants (north-eastern, NE; north-western, NW; south- selected, and the dating procedure was repeated with the eastern, SE; south-western, SW) as divided by the geographical following parameters: collapse; num_time_guesses = 5 and coordinates 40 N/5 W; northern Africa; and Europe (exclud- num_restarts = 5. Cross-validation suggested 100,000 as the ing Iberia) plus western Asia (Table 1). best smoothing parameter. Branching order and branch lengths from 1000 Bayesian trees (500 trees from each run), sampled every 5000 generations after stationarity, were anal- Phylogenetic analyses ysed to obtain means and standard deviations of clade ages The plastid and nuclear ribosomal DNA (nrDNA) sequence (Hughes & Eastwood, 2006). As recommended in the r8s data sets were analysed using maximum parsimony (MP) and manual, we pruned the extra outgroup (Callitriche brutia)in Bayesian inference (BI) approaches. Parsimony analyses were order to infer the root node of the real tree. performed in paup* (Swofford, 2002) using a heuristic search replicated 1000 times with random taxon-addition sequences, RESULTS tree bisection–reconnection (TBR) branch swapping, the options MulTrees and Steepest Descent in effect, and holding Plastid haplotypes 100 trees at each step. To evaluate the internal support of each clade, 100 bootstrap replicates (Felsenstein, 1985) were The aligned trnS-trnG and trnK-matK Antirrhinum sequences performed using equal weights, the TBR swapping algorithm were 672 and 1350 base pairs long, respectively. We detected 51 with 10 random additions of taxa per bootstrap replicate, and haplotypes (with no gap recoded), of which 37 are exclusive to 100 trees held at each step. The appropriate model of single accessions (Table 1). The 51 haplotypes were connected in nucleotide substitution for BI was determined by the hierar- a star-like network with no more than seven inferred mutational chical likelihood ratio test (hLRT) and the Akaike information changes to connect any pair of haplotypes (Fig. 2). Outgroup criterion (AIC) implemented using MrModeltest ver. 1.1b (Misopates, Gambelia, Acanthorrhinum) sequences were discon- (Posada & Crandall, 1998; Nylander, 2002). Bayesian inference nected. A low number (27) of missing haplotypes (extinct or not analyses were conducted in MrBayes ver. 3.2.1 (Ronquist & found) were inferred to connect all sampled haplotypes (51). Huelsenbeck, 2003). Two identical searches with three million The interior haplotype 1 is shared by samples of five species generations each (chain temperature = 0.2; sample fre- (A. braun-blanquetii, A. graniticum, A. linkianum, A. litigiosum, quency = 100) were performed. In both runs, probabilities A. lopesianum) from three geographical areas of Iberia (SE, NE, converged on the same stable value, approximately after NW). Haplotype sharing was also observed in samples of the generation 500,000 in the plastid and 300,000 in the nuclear following species groups: A. charidemi–A. mollissimum (haplo- analyses. A 50% majority-rule consensus tree was calculated type 37 from SE Iberia), A. grosii–A. meonanthum (haplotype 30 using the sumt command to yield the final Bayesian estimate of from NW Iberia), A. latifolium–A. majus–A. molle (haplotype 11 each phylogeny. We used posterior probability (PP) as an from NE Iberia), A. majus–A. molle (haplotype 12 from NE estimate of robustness. Iberia) and A. pulverulentum–A. subbaeticum (haplotype 25 from NE and SE Iberia). The eight major haplotype clades (formed by two or more Dating lineage divergence haplotypes) depicted in the network analysis are not necessar- No reliable fossil data of Antirrhinum and relatives (the tribe ily related to species assignation (Table 1, Fig. 2). Interestingly, Antirrhineae) are known. We therefore used the estimated age the limited exclusiveness of haplotypes associated with species (38–48 Ma) of the split between the Plantaginaceae and names contrasts with the geographical distribution of haplo- Antirrhinum as obtained by Wikstro¨m et al. (2001). As types. SE Iberia harbours the highest number of haplotypes outgroup sequences could not be reliably aligned for the ITS (21), followed by NE Iberia (18), NW Iberia (5), N Africa (4), and trnS-trnG regions, we used the plastid trnK-matK data set Europe excluding Iberia (4), and SW Iberia (3). Admittedly, to estimate lineage divergence. Tree topology and branch the two areas (SE and NE Iberia) with a high number of lengths were estimated for this data set under the BI approach. haplotypes were sampled more intensively, albeit they also We used the same parameters as above. Probabilities con- contain a higher number of species. Phylogeographical results verged on the same stable value after approximately generation suggest a complex pattern in NE Iberia, given the number of 500,000 in the two Bayesian runs. unrelated haplotype clades (I, II, V, VI), of which one (V)

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Figure 2 Statistical parsimony network of 51 plastid haplotypes found in Antirrhinum, as deﬁned on the basis of plastid trnS-trnG/trnK- matK sequences. Lines represent single mutational steps; black, small circles are inferred haplotypes not found in any sample. Colours in the inset refer to geographical areas (see text). Roman numbers indicate major plastid lineages with two or more haplotypes. Haplotype distribution across species is as follows: A. australe (17, 42, 43); A. braun-blanquetii (1, 7); A. charidemi (37, 49); A. cirrhigerum (15); A. controversum (34, 35, 36, 40); A. graniticum (1, 29); A. grosii (30, 31); A. hispanicum (38, 39, 41); A. latifolium (11); A. linkianum (1, 8); A. litigiosum (1, 5); A. lopesianum (1, 6); A. majus (9, 10, 11, 12); A. meonanthum (29, 30); A. microphyllum (24, 26); A. molle (11, 12); A. mollissimum (37); A. pertegasii (4); A. pulverulentum (3, 25, 27, 28, 50, 51); A. sempervirens (2, 13, 14); A. siculum (21, 22, 23); A. subbaeticum (25, 32, 33); A. tortuosum (16, 18, 19, 20); and A. valentinum (44, 45, 46, 47, 48) (see Table 1). contains four samples from two more geographical areas and contained at least one IUPAC symbol, reﬂecting more than a haplotypes (25, 29) shared by two different geographical areas single nucleotide. Fifty-nine electropherograms of the 87 (Table 1, Fig. 2). Clade III also reveals a certain phylogeo- Antirrhinum accessions displayed double peaks of similar graphical complexity, with an association of haplotypes from height (nucleotide additivities). The number of additivities per SW Iberia, Europe and N Africa. Cohesiveness is, in contrast, accession varied from one to 16 (Table 1). Although it could observed in 18 of 21 haplotypes from SE Iberia (Fig. 2). not be determined, in some cases, whether double-peak patterns were the result of sequencing artefacts, equimolar proportions of alternative nucleotide peaks are interpreted in ITS sequence variation many accessions as the presence of different ITS copies. We The ITS sequences (ITS-1+5.8S+ITS-2) ranged between 586 observed a limited number of sequencing artefacts versus a and 603 base pairs in Antirrhinum, resulting in an aligned general pattern of multiple ITS copies contained in single matrix of 145 variable and 80 parsimony-informative charac- Antirrhinum samples because: (1) two alternative nucleotides ters. All accessions are considered ITS functional copies, rather at the same site were widely present across 59 of the 87 than pseudogenes, because they are similar not only to other accessions; (2) most ambiguous nucleotides of these 59 Antirrhineae species (Vargas et al., 2004), but also to other accessions were at the 80 parsimony-informative positions; plant taxa. The number of variable/parsimony-informative (3) only two of the 164 sites of the 5.8S showed additivities, as sites was distributed as follows: 80/42 in ITS-1, 2/2 in 5.8S and expected from a highly conserved region; (4) previous clones 63/36 in ITS-2. In our data set, 105 of the 145 variable sites of ITS products of A. litigiosum (population 2) revealed

1304 Journal of Biogeography 36, 1297–1312 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd Geographical speciation in Antirrhinum alternative nucleotides at seven additive sites (Vargas et al., divergence between the Plantaginaceae–Scrophulariaceae and 2004); and (5) controlled hybrids between A. pulverulen- the tribe Antirrhineae (Wikstro¨m et al., 2001), the divergences tum · A. barrelieri displayed complex additivity patterns, of lineages of Antirrhineae (21.49 ± 4.27 Ma) and Antirrhi- including the maintenance of some of the parental additivities num (4.10 Ma to present; error term truncated) probably took and the generation of new ones (data not shown). Multiple ITS place in the Miocene and Pliocene–Pleistocene, respectively. copies in a single individual can be derived by DNA mutation Given the large number of trees with remarkably different in the ITS region (ITS divergence) or by the merging of topologies, it is not possible to propose a single phylogenetic different nrDNA copies into single genomes (hybridization) hypothesis, and thus to estimate divergence times within followed by failure in concerted evolution (Arnheim, 1983; Antirrhinum (Fig. 4). All analyses are, however, congruent with Whittall et al., 2000). The fact that accessions from isolated the differentiation of current Antirrhinum post-dating the populations (A. grosii, A. lopesianum, A. siculum) displayed no Miocene (Fig. 4). or lower additivities than those from populations in areas with a high number of Antirrhinum species (A. controversum 6, DISCUSSION A. litigiosum 2, A. majus 8, A. mollissimum 2, A. pertegasii 2, A. tortuosum 2) led us to conclude that it is hybridization that Evidence for extensive hybridization in Antirrhinum is primarily responsible for this nucleotide additivity pattern (Table 1). The phylogenetic results show a limited number of monophyletic groups of Antirrhinum accessions related to sections and species. High levels of homoplasy (correspondence of charac- Phylogenetic analyses ters acquired as the result of parallel, reversal or convergent The number of variable/parsimony-informative characters was evolution), lineage sorting (persistence of ancestral polymor- 55/26 in the trnS-trnG and trnK-matK sequence matrix of phism through speciation events) and reticulation (non- Antirrhinum accessions. The models of substitution selected by hierarchical gene transfer) can produce similar phylogenetic MrModeltest (Posada & Crandall, 1998; Nylander, 2002) were results (Linder & Rieseberg, 2004). The parsimony estimates of GTR+G (trnS-trnG), GTR (trnK-matK) and GTR+I+G (ITS). CI (0.94) and RI (0.97) obtained from the parsimony analysis Both BI and MP analyses of the plastid and ITS sequence data of plastid sequences illustrate that the levels of homoplasy produced a low number of well-supported groups (Fig. 3; found in our data set reach similarly low values to those of results not shown for ITS). The plastid analysis retrieved other angiosperm groups (A´ lvarez & Wendel, 2003). Differ- Antirrhinum accessions as monophyletic (Figs 3 & 4). The BI entiating between hypotheses of lineage sorting and hybrid- tree based on plastid sequences revealed only 10 species groups ization is, however, extremely difficult because both of these supported by posterior probability values equal to or higher processes can generate incongruent phylogenetic patterns, and than 95 (Fig. 3). The MP analysis of plastid sequences rendered thus additional sources of evidence are needed (Linder & 237 most-parsimonious trees of 142 steps [consistency index Rieseberg, 2004). Our results of plastid and ITS sequence (CI) = 0.94; retention index (RI) = 0.97] and even lower levels polymorphism are better explained by the historical hypothesis of support, with only four species groups in clades with of extensive hybridization in Antirrhinum. The ITS region is a bootstrap values > 75% (Fig. 3). Irrespective of support values, multicopy DNA region, and the presence of two or more no monophyletic groups including all conspecific samples of ancient copies may be obscured by processes of concerted Antirrhinum were retrieved in the two analyses, except for the evolution, which operate by recombinational mechanisms two A. pertegasii accessions. In contrast, numerous clades occurring during meiosis (Whittall et al., 2000). In contrast, include exclusively or primarily accessions from the six failure in ITS copy fixation allows the possibility of detecting geographical areas (Figs 2 & 3). All major plastid (species) additive patterns (both parental ribotypes present), which have lineages include samples from SE or NE Iberia. Neither MP nor been historically related to polyploidy and homoploid hybrid- BI analyses of ITS sequences, however, found major accession ization (Arnheim, 1983; Sang et al., 1995; Vargas et al., 1999; groupings; instead a large, unresolved polytomy was retrieved Whittall et al., 2000). Nucleotide additivity sites may consti- (results not shown), similar to previous findings using a more tute evidence for reticulation under the assumption that limited sample (Vargas et al., 2004). The high number of ITS polymorphism has resulted from the merging of divergent ITS most-parsimonious trees (91,100) and low consistency index repeats in a single genome. Our ITS sequence analysis strongly (0.57) and retention index (0.77) also revealed a serious failure supports multiple events of acquisition of different ITS copies. to obtain a cladogenetic pattern in Antirrhinum. In fact, the analysis herein performed using an extended sample (87 accessions from 24 of the 25 species) fits previous predictions of extensive failure in nucleotide fixation across Lineage divergence dating Antirrhinum populations (Vargas et al., 2004). The assumption of the hypothesis of equivalent rates of Multiple ITS copies in single samples displaying additive sequence evolution across lineages was rejected (v = 218.37, patterns have been widely related in literature to recent d.f. = 96). Estimates of divergence times using the PL method hybridization in angiosperms (Arnheim, 1983; Fuertes-Aguilar are shown in Fig. 4. Our results indicate that, after the et al., 1999; Bailey et al., 2003; Nieto-Feliner & Rossello´, 2007).

Journal of Biogeography 36, 1297–1312 1305 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd P. Vargas et al.

Figure 3 Phylogenetic analysis of plastid trnS-trnG/trnK-matK sequences of 24 Antirrhinum species based on the 50% majority consensus tree of the Bayesian inference analysis. Numbers above branches are posterior probability values. Numbers below branches show both branch agreement with the strict consensus tree and bootstrap support > 50%. One dash (–) below branches indicates bootstrap support < 50%, whereas two dashes (––) indicate disagreement between the maximum parsimony (MP) strict consensus tree and the BI tree. Sample coding as in Table 1. Geographical abbreviations: SE, south-eastern Iberia; SW, south-western Iberia; NE, north-eastern Iberia; NW, north-western Iberia; NA, northern Africa; E, Europe (excluding Iberia) and western Asia.

1306 Journal of Biogeography 36, 1297–1312 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd Geographical speciation in Antirrhinum

Figure 4 Chronogram based on the Bayesian consensus tree and the penalized likelihood (PL) analysis of the trnK-matK sequences. Branch lengths represent millions of years (Ma). A previous estimated date of the Plantaginaceae–Scrophulariaceae and the tribe Antirrhineae split at 38–48 Ma (Wikstro¨m et al., 2001) was used to implement the analysis. Sample coding as in Table 1. A vertical, grey strip indicates the establishment of the current Mediterranean climate in the Mediterranean basin.

Journal of Biogeography 36, 1297–1312 1307 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd P. Vargas et al.

Indeed, the lack of monophyly of plastid haplotypes into species Pyrenean populations has been historically predominant in (Jimeńez et al., 2005b; see below) and disagreement between Antirrhinum, given that self-incompatibility in the majority of plastid (Fig. 3) and nuclear (Vargas et al., 2004; Langlade et al., Antirrhinum species favours hybridization (Sutton, 1988; Ma- 2005) tree-based topologies for some clades further support a teu-Andre´s & de Paco, 2006; P. Vargas et al., data not shown). severe failure to retrieve cladogenetic processes. Although broad Molecular markers can therefore provide unambiguous reticulation is difficult to demonstrate conclusively in all ‘footprints’ of ancient hybridization events (haplotype diver- instances, genetic and morphological results from Antirrhinum sity significantly related to geography, but unrelated to species accumulated in recent years clearly fit into the hybridization boundaries) and recent hybridization contacts (limited nucle- model previously described in angiosperms because of: (1) the otide additivity homogenization). However, identifying which difficulties in recovering any synapomorphy in cladistic analyses ancestral lineages (taxa) have hybridized is not possible, given based on a small number of intermediate morphological the complicated pattern of our results. The question remains of characters (Sutton, 1988; Vargas et al., 2004; see also McDade, whether hybridization is the only process historically involved 1990); (2) the low resolution in parsimony analyses of nrDNA in Antirrhinum speciation. sequences (this paper; Vargas et al., 2004; see also Soltis et al., 2008); (3) the significant conflict between ITS, plastid and Pleistocene divergence of Antirrhinum lineages morphological results (Vargas et al., 2004; Jimeńez et al., 2005b; see also McDade et al., 2005); (4) the additivity patterns from Phylogeography explores the lineage relationships among dominant (Random Amplification of Polymorphic DNA; populations of the same (or closely related) species in a Jimeńez et al., 2005a) or co-dominant (ITS sequences, Table 1; geographical context (Avise, 2000). Antirrhinum, with 24 see also Soltis et al., 2008) molecular markers; (5) the close species sampled in this study, displays low levels of genetic relationship between geography and haplotype distribution diversity. In fact, haplotype number (51) and one single (Fig. 2; see also Fuertes-Aguilar & Nieto-Feliner, 2003); and (6) network retrieved in the phylogeographical analysis (Fig. 2) the high number of ITS additivities in overlapping geographical parallel phylogeographical results at the populational level of areas of certain species (Fig. 1, Table 1; see also Fuertes-Aguilar particular species (Albadalejo et al., 2005; Koch et al., 2006; & Nieto-Feliner, 2003). Besnard et al., 2007). The fact that 51 haplotypes have been The viability of interspecific hybrids further supports a found in Antirrhinum and only 27 have been inferred to be scenario of extensive hybridization (Baur, 1932; Mather, 1947). missing (extinct or not found) per se indicates: (1) that our With the exception of A. siculum, all the species are interfertile sample is suitable for defining haplotype clades; (2) close (Langlade et al., 2005). The ease with which consecutive species relatedness; and (3) short divergence times. generations can be obtained in artificial crossings indicates a Hypothetical pre-Pliocene differentiation of Antirrhinum, lack of genetic barriers between species (Rothmaler, 1943; based on ITS sequence variation (Vargas et al., 2004) and

Thompson, 1988; Xue et al., 1996). Indeed, F1 generations Rothmaler’s (1956) arguments, is not supported by our between species displaying the most extreme phenotypes have calibrated plastid phylogeny. The PL clock method using been used to quantify leaf (Langlade et al., 2005) and flower trnK-matK sequences is largely congruent with a differentiation (Whibley et al., 2006) gene expression. The importance of process of extant Antirrhinum lineages since the Pliocene hybridization in Antirrhinum has also been identified by the (Fig. 4). These estimates of divergence times are consistent description of ‘unilateral hybridization’, which can be made with previous estimates based on CYCLOIDEA-like genes only in one direction, namely, when the self-fertile species of the (Gu¨bitz et al., 2003). The increasing number of studies of pair is used as female parent and the self-incompatible one as Mediterranean plants reveals evidence of active differentiation male (Harrison & Darby, 1955). Natural hybrids have been in the Pliocene and Pleistocene, as coincident with the onset of easily detected in the field where two or more species the Mediterranean climate (Conti, 2007; Guzmań & Vargas, occasionally meet (Rothmaler, 1956). Our intensive fieldwork 2008, for Cistus). However, we add a cautionary note that our studies over the last 10 years have contributed to the finding estimated divergence times were calibrated on an estimated time of four new interspecific hybrids in addition to the seven of divergence between Plantaginaceae and Antirrhinum from previously named (J. Guëmes & P. Vargas, unpublished data). Wikstro¨m et al. (2001). Topological irresolution and short Hybridization processes may also have contributed to historical branch length at the base of plastidial and nuclear phylogenies taxonomic disagreement (Sutton, 1988). Individuals of a hybrid are again congruent with recurrent hybridization but also with a zone involving A. majus and a taxonomically disputable entity slow rate of sequence change and with Antirrhinum radiation. (A. striatum) display intermediate and novel flower colour This could be interpreted given a large number of species (25) phenotypes found only in the Pyrenees (Whibley et al., 2006). originating in a short period of time (Fig. 4). Our sequence analyses are congruent with hybridization processes in a large area in the eastern Pyrenees, where two (I, A geographical pattern of differentiation in eastern II) haplotype lineages meet (Figs 1 & 2), and individual samples Iberia contain a considerable number (2–10) of ITS additivities (A. latifolium 1 and 2, and A. majus 1, 3, 5 and 6; Table 1). The haplotype distribution and plastid DNA (cpDNA) We suggest that this pattern of interfertility observed now in network (Fig. 2) give clear evidence that geography has been

1308 Journal of Biogeography 36, 1297–1312 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd Geographical speciation in Antirrhinum the major factor in Antirrhinum differentiation. In contrast to resulted in the increase of allozyme gene alleles (Mateu-Andre´s disassociation between haplotypes and species assignation, & Segarra-Moragues, 2000) and haplotype diversity (Table 1). geographical cohesion is observed. South-eastern Iberia Similar morphological phenotypes, as a result of further appears to be a main centre of differentiation, as revealed by geographical isolation of populations in restricted areas the highest figures of haplotypes/haplotype clades (21/4), (Fig. 1a), may be the cause of the undisputable taxonomic closely followed by NE Iberia (18/4), and then by Europe recognition of A. valentinum and A. subbaeticum. In fact, excluding Iberia (4/2), N Africa (4/1), NW Iberia (5/1) and genetic phenotypes (RAPDs) unequivocally discriminated SW Iberia (3/1) (Table 1/Fig. 2). In addition, eastern Iberia populations into these two species (Jimeńez et al., 2005a). harbours the highest number of species (15 of the 25 Optimal ecological conditions may also have been impor- Antirrhinum species), indicating a relatively long history of tant in Antirrhinum evolution (Whibley et al., 2006). In Antirrhinum differentiation and speciation since the establish- addition to isolated rocky habitats in the western Mediterra- ment of the Mediterranean climate. These results were not nean region, territorial bees such as Anthidium spp. pollinating unexpected. Habitat requirements (rocky soils) for most the characteristic personate flower of Antirrhinum species Antirrhinum species are found to be particularly abundant in (Glover & Martin, 1998; Torres et al., 2003; Vargas, 2007) the mountains of eastern Iberia. Within the Mediterranean should be further explored to infer the most recent processes of region (Myers et al., 2000), SE Iberia is a hotspot for diversity disruptive selection and speciation. as manifested not only by the high number of species and high level of endemism (Sainz Ollero & Moreno Saiz, 2002), but ACKNOWLEDGEMENTS also by the accumulation of divergent lineages within different angiosperm groups (Quercus, Lumaret et al., 2005; Hedera, The authors thank Alan Forrest, Andrew Hudson, Yvette Valca´rcel et al., 2003; Phlomis, Albadalejo et al., 2005; Arena- Erasmus, Pauline Ladiges, Phil Garnock-Jones and an anon- ria, Valca´rcel et al., 2006). ymous referee for detailed comments on the manuscript; Limited distributions of seven species (Fig. 1b), including E. Cano for laboratory assistance; and G. Bacchetta, I. Marques, recognition of four endangered species (Banãres et al., 2003), S. Ortiz and F. Amich for plant materials. This research was additionally suggests that geography has had an important role supported by two Spanish projects (REN2002-2434, and in Antirrhinum evolution. Geographical speciation may have CGL2005-05471-C04-01). taken place in Iberian mountains, followed by secondary contacts, with range expansions and contractions reflecting REFERENCES climate fluctuations in the Quaternary (Hewitt, 2000). The mountainous eastern Iberia offers a large area within which to Albadalejo, R.G., Fuertes-Aguilar, J., Aparicio, A. & Nieto- infer the evolutionary causes of either historical or recent Feliner, G. (2005) Contrasting nuclear-plastidial phylo- isolation of two endangered species (A. subbaeticum, A. genetic patterns in the recently diverged Iberian Phlomis valentinum) (Fig. 1b). Chorological, phylogenetic, phylogeo- crinita and P. lychnitis lineages (Lamiaceae). Taxon, 54, graphical, population genetic, morphological and ecological 987–998. data are consistent with the following scenario. 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Journal of Biogeography 36, 1297–1312 1311 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd P. Vargas et al.

BIOSKETCH

Pablo Vargas (Royal Botanic Garden of Madrid) and co-authors study various questions about the micro- and macro-evolutionary mechanisms responsible for evolutionary changes in the tribe Antirrhineae and the genus Antirrhinum. An integrative view and methodology, including approaches in taxonomy, molecular phylogenetics, phylogeography, genetics and ecology, are used to connect all sources of variation and address explicit evolutionary hypotheses (http://www3.rjb.csic.es/snapdragons/).

Editor: Pauline Ladiges

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.

1312 Journal of Biogeography 36, 1297–1312 ª 2009 The Authors. Journal compilation ª 2009 Blackwell Publishing Ltd