The biogeographical history of the cosmopolitan genus Ranunculus L. (Ranunculaceae) in the temperate to meridional zones

B. Gehrke

A phylogeographical analysis of Ranunculus platanifolius, a typical European subalpine tall-herb species, indicates the existence of two main genetic lineages based on amplified fragment length polymorphism (AFLP) markers. One group comprises populations from the Balkan Peninsula and the south-eastern Carpathians and the other includes the remaining part of the range of the species, encompassing the western Carpathians, Sudetes, Alps, Pyrenees and Scandinavia. The main phylogeographical break observed in this species runs across the Carpathians and separates the main parts of this range (western and south-eastern Carpathians), supporting a distinct glacial history of populations in these areas. The high genetic similarity of the Balkan Peninsula and south-eastern Carpathian populations could indicate a common glacial refugium for these contemporarily isolated areas of species distribution. The western and northern part of the species range displays an additional weak differentiation into regional phylogeographical groups, which could have been shaped by isolation in glacial refugia or even by a postglacial isolation. The observed weak phylogeographical structure could also be linked with ecological requirements, allowing survival along streams in relatively low, forested mountain ranges. © 2013 The Linnean Society of London

There is considerable controversy concerning the fate of Alpine plants during Pleistocene glaciations. While some studies have found evidence for nunatak survival, others have explained the present genetic patterns by survival only in peripheral refugia. We investigated 75 populations of high alpine Ranunculus glacialis from its entire Alpine distribution. Phylogeographical analyses of AFLP data revealed four groups of populations. Two of them, located in the western Alps, were genetically isolated from each other and from the eastern groups, whereas the two eastern Alpine groups were genetically more similar to each other. This suggests longer isolation and/or lower levels of gene flow in the two western groups. As all groups are close to, or overlap with, presumed glacial refugia, invoking glacial survival on nunataks is unnecessary to explain the present genetic pattern. Similar to the phylogeographical patterns of R. glacialis, the previously investigated alpine Phyteuma globulariifolium and Androsace alpina, which are also confined to siliceous bedrock, showed strong geographical affinities to peripheral refugial areas and there were large-scale congruencies in the location of these refugia for all three species. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81, 183–195.

Although many species have similar total distributional ranges, they might be restricted to very different habitats and might have different phylogeographical histories. In the European Alps, our excellent knowledge of the evolutionary history of silicate-dwelling (silicicole) plants is contrasted by a virtual lack of data from limestone-dwelling (calcicole) plants. These two categories exhibit fundamentally different distribution patterns within the Alps and are expected to differ strongly with respect to their glacial history. The calcicole Ranunculus alpestris group comprises three diploid species of alpine habitats. Ranunculus alpestris s. str. is distributed over the southern European mountain system, while R. bilobus and R. traunfellneri are southern Alpine narrow endemics. To explore their phylogenetic relationships and phylogeographical history, we investigated the correlation between information given by nuclear and chloroplast DNA data. Analyses of amplified fragment length polymorphism fingerprints and matK sequences gave incongruent results, indicative for reticulate evolution. Our data highlight historical episodes of range fragmentation and expansion, occasional long-distance dispersal and on-going gene flow as important processes shaping the genetic structure of the group. Genetic divergence, expressed as a rarity index (‘frequency-down-weighted marker values’) seems a better indicator of historical processes than patterns of genetic diversity, which rather mirror contemporary processes as connectivity of populations and population sizes. Three phylogeographical subgroups have been found within the R. alpestris group, neither following taxonomy nor geography. Genetic heterogeneity in the Southern Alps contrasts with Northern Alpine uniformity. The Carpathians have been stepwise-colonised from the Eastern Alpine lineage, resulting in a marked diversity loss in the Southern Carpathians. The main divergence within the group, separating the ancestor of the two endemic species from R. alpestris s. str., predates the Quaternary. Therefore, range shifts produced by palaeoclimatic oscillations seem to have acted on the genetic structure of R. alpestris group on a more regional level, e.g. triggering an allopatric separation of R. traunfellneri from R. bilobus.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Molecular Phylogenetics and Evolution 58 (2011) 4–21 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev The biogeographical history of the cosmopolitan genus Ranunculus L. (Ranunculaceae) in the temperate to meridional zones Khatere Emadzade a, Berit Gehrke b, H. Peter Linder c, Elvira Hörandl a,⇑ a Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria Department of Botany, University of Cape Town, Private Bag X3, 7701 Rondebosch, South Africa c Institute for Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland b a r t i c l e i n f o Article history: Received 18 November 2009 Revised 12 October 2010 Accepted 3 November 2010 Available online 13 November 2010 Keywords: Ranunculus Molecular phylogenetics Biogeographical history Dispersal Vicariance a b s t r a c t Ranunculus is distributed in all continents and especially species-rich in the meridional and temperate zones. To reconstruct the biogeographical history of the genus, a molecular phylogenetic analysis of the genus based on nuclear and chloroplast DNA sequences has been carried out. Results of biogeographical analyses (DIVA, Lagrange, Mesquite) combined with molecular dating suggest multiple colonizations of all continents and disjunctions between the northern and the southern hemisphere. Dispersals between continents must have occurred via migration over land bridges, or via transoceanic long-distance dispersal, which is also inferred from island endemism. In southern Eurasia, isolation of the western Mediterranean and the Caucasus region during the Messinian was followed by range expansions and speciation in both areas. In the Pliocene and Pleistocene, radiations happened independently in the summerdry western Mediterranean–Macaronesian and in the eastern Mediterranean–Irano-Turanian regions, with three independent shifts to alpine humid climates in the Alps and in the Himalayas. The cosmopolitan distribution of Ranunculus is caused by transoceanic and intracontinental dispersal, followed by regional adaptive radiations. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Ranunculus s.str. is a cosmopolitan genus with approximately 600 species (Tamura, 1993, 1995) and the largest genus in Ranunculaceae. Ranunculus is distributed on all continents and it has a worldwide distribution from the Tropics to the arctic and subantarctic zones. The genus is especially species-rich in temperate to meridional zones (e.g., Ovczinnikov, 1937; Iranshahr et al., 1992; Whittemore, 1997). In the tropical areas, species are restricted to high mountain areas (e.g. African species; Tamura, 1993, 1995). Species of Ranunculus are established in a variety of wet to dry habitats from the lowland to high alpine zones and show several morphological adaptations to different habitats (Paun et al., 2005). In mountain areas, endemism contributes to the considerable species diversity, but in lower altitudes widespread species are also quite common. Ranunculus shows different levels of polyploidy, which is sometimes connected to apomixis (Hörandl et al., 2005). Monophyly of Ranunculus has been assumed by previous molecular phylogenetic studies (Hoot, 1994; Johansson, 1995, 1998; Ro et al., 1997; Hörandl et al., 2005; Paun et al., 2005; Lehnebach et al., 2007; Gehrke and Linder, 2009; Hoffmann et al., 2010; Emadzade et al., 2010). Previous studies (using cpDNA restriction sites, ⇑ Corresponding author. Fax: +43 1 4277 9561. E-mail address: elvira.hoerandl@univie.ac.at (E. Hörandl). 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.11.002 Johansson, 1998; ITS sequences, Hörandl et al., 2005; matK/trnK plus ITS, Paun et al., 2005; Lehnebach, 2008; Gehrke and Linder, 2009; Hoffmann et al., 2010) showed that the core Ranunculus clade was subdivided into several well-supported clades that corresponded to widespread ecological groups (e.g., wetland and aquatic species) or to regional geographical groups (e.g., in the European mountain system; Hörandl et al., 2005; Paun et al., 2005). Biogeographical studies focusing on certain areas suggested multiple colonizations of Africa (Gehrke and Linder, 2009) and of the Arctic (Hoffmann et al., 2010). However, the biogeographical processes that have shaped the global distribution of buttercups are still not well understood. The frequently observed intercontinental disjunctions in earlier studies could be due to wide distributions of the ancestors that have been separated via geographical barriers, followed by allopatric speciation and diversification (vicariance). Alternatively, the lineages within the clades had the ability for dispersal via seeds or propagules to new areas, followed by speciation and adaptation to new habitats. Endemism on oceanic islands like Hawaii, Juan Fernandez Islands, and Macaronesia is another indication for the high ability of buttercups for longdistance dispersal (LDD), speciation and rapid adaptation to new habitats. A northern hemispheric origin followed by vicariance and transoceanic dispersal has shaped the distributional patterns in Ranunculeae (Emadzade and Hörandl, in press). These genera, however, are not as diversified and widely distributed as Ranunculus Author's personal copy K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 s.str.; some of them are monotypic and regional endemics. In contrast, Ranunculus s.str., shows not only the ability for long-distance dispersal to new areas, but also a potential for adaptive radiations (Lockhart et al., 2001; Paun et al., 2005). The origin of Ranunculus probably dates back to the late Oligocene, and was followed by several waves of diversification until the Quaternary (Paun et al., 2005; Hoffmann et al., 2010; Emadzade and Hörandl, in press). The high number of species and endemism, the global distribution, and the observed temporal and spatial patterns make this genus interesting for studying historical biogeography. However, previous studies focused only on certain regions (Mediterranean, Paun et al., 2005; Africa, Gehrke and Linder, 2009; Arctic, Hoffmann et al., 2010) and did not apply analytical tools of biogeography. The biogeographical history of related genera has been presented elsewhere (Hörandl and Emadzade, in press). A comprehensive biogeographical analysis of the cosmopolitan genus Ranunculus s.str. based on a worldwide sampling has been so far missing. Based on a molecular phylogenetic reconstruction (Figs. 1a and 1b), we focus here on a species-rich clade comprising mainly species of the temperate to the meridional zones (Fig. 1a, clades V–IX). We did not attempt a reconstruction of the biogeographical history of Ranunculus as a whole, because, at first, the backbone phylogeny is not well resolved, and relationships of big clades (I–IX) to each other are not well supported (Figs. 1a and 1b); second, the clades with meridional-temperate (V–IX) species showed a better resolution compared to the high alpine, arctic or wetland groups (Fig. 1b, clades I–IV). Previous molecular dating approaches (Paun et al., 2005; Hoffmann et al., 2010; Emadzade and Hörandl, in press) suggested origin and diversification of the meridional-temperate clades already in the Miocene. Because of the age and the southern distribution, the spatial–temporal diversification in these clades was not so much influenced by range fluctuations and extinctions due to Quaternary glaciations. The meridional to temperate clades comprise species from all continents (except Antarctica), which allows for the analysis of intercontinental disjunctions, dispersal, and vicariance events between continents in a global framework. Previous phylogenetic studies suggest that the temperate zone was the source area for both subtropical–tropical and arctic species (Gehrke and Linder, 2009; Hoffmann et al., 2010). The biogeographical processes in the meridional to temperate zones are therefore of crucial importance for understanding the biogeographical history of the genus. The species-richness in these areas further raises the question whether intracontinental dispersal and regional radiations have played a role for the diversity and wide distribution of the genus. For this question, the biogeographical patterns in southern Eurasia can serve as a model system for other continents. This ‘‘ancient Tethyan area’’ (Takhtajan, 1986) is of special interest for biogeographical questions because of its complex geological and climatic history. In the Mediterranean area, sea-level fluctuations, including desiccation and later re-flooding of the Mediterranean sea, establishment of a summer-dry climate and the uplift of mountain chains have caused both geographic and eco-climatological differentiation processes in flowering plants (Thompson, 2005; Lo Presti and Oberprieler, 2009). The direct geographical connection of the Mediterranean to the Irano-Turanian region, and the continuation of the European Alpine system in the Central Asian mountain chains provide migration routes and have formed a distinct biogeographical area (‘‘ancient Tethyan area’’ sensu Takhtajan, 1986). In contrast, the alpine–arctic (Fig. 1b, clades I, II and IV) and the wetland clades (clade III) have been influenced by reticulate evolution, hybridization and high frequencies of polyploids which is problematic for tree-based biogeographical analyses because of a non-hierarchical structure of data (Lockhart et al., 2001; Hörandl et al., 2005, 2009). In clades I–III, our analyses confirmed 5 geographical patterns of previous studies (Hörandl et al., 2005; Paun et al., 2005): clades I and II comprise mainly European alpine species, while clade III consists of widespread wetland or aquatic species. The evolutionary history of the alpine-arctic clade (Fig. 1b, clade IV) will be presented elsewhere (Hörandl and Emadzade, in press). Molecular phylogenetic data, including molecular age estimates, provide a strong hypothesis for understanding the biogeographical history of the temperate–meridional species. We use here the information from previous molecular dating studies (Paun et al., 2005; Hoffmann et al., 2010; Emadzade and Hörandl, in press) to provide a comprehensive hypothesis of the history of Ranunculus in the meridional to temperate zones in a global context. We use biogeographical analyses to (1) develop hypotheses for the spatial distribution of buttercups in the context of the geological history of the different continents, (2) to investigate the main migration routes between continents and areas of diversity, and (3) to reconstruct the main factor(s) shaping the modern distribution of the genus, including the relative role of long-distance dispersal and vicariance. Additionally, we identify the main processes that have caused the modern distribution and diversity of taxa in greater detail in the ‘‘Tethyan’’ clade, Fig. 1a, clade IX) comprising species from the whole Mediterranean–Macaronesian area, the Circumboreal area, the Irano-Turanian region, Central Asia, and the Himalayas. 2. Materials and methods 2.1. Plant material We sampled 185 species of Ranunculus s.str. (Tamura, 1993, 1995) and 20 species of allied genera to develop a basic phylogenetic framework. This collection covers more than one third of the buttercups from all continents except Oceania and Antarctica from where samples were not available. However, only few buttercup species occur in these areas, and lack of samples from these areas does not strongly influence the distribution patterns in the temperate to meridional zones. The sampling further covers all taxonomic sections sensu Tamura (1995). Anemone and Isopyrum were used as outgroup taxa (Emadzade et al., 2010). A nuclear marker (the ITS region of the nuclear ribosomal DNA) and two chloroplast markers (matK/trnK) were obtained from 71 new species and combined with data from previous studies (Hörandl et al., 2005; Paun et al., 2005; Gehrke and Linder, 2009; Hoffmann et al., 2010). The psbJ–petA region was newly sequenced for all species. We used only samples for which sequences of all markers were available. Voucher information and GenBank accession numbers are provided in Table 1. 2.2. DNA extraction, amplification, and sequencing Total genomic DNA from silica-dried or herbarium material was extracted according to the CTAB-procedure (Doyle and Doyle, 1987) with some modifications (Tel-Zur et al., 1999). The whole internal transcribed spacer region (ITS, including ITS1, the 5.8 gene, ITS2) was amplified as a single piece with primers ITS 18sF and ITS 26sR (Gruenstaeudl et al., 2009) or in the case of degraded DNA from poor quality herbarium tissue, in two pieces with additional primers (ITS 5.8sF and ITS 5.8sR) as internal primers (Gruenstaeudl et al., 2009). Sequencing of the matK/trnK region was performed according to the protocol described by Paun et al. (2005). Amplification of the non-coding PsbJ/PetA region was carried out as a single piece in all samples by using primers of Shaw et al. (2007). PCR was performed in 23 ll reactions containing 20 ll 1.1 Reddy Mix PCR Master Mix (including 2.5 mM MgCl2; ABgene, Epsom, UK), Author's personal copy 6 B, meridional-temperate clade IX VIII VII A, alpine- arctic- wetland clades VI V IV III II I allied genera MP R oxyspermus R psilostachys R rumelicus R sprunerianus R argyreus R damascenus R macrorrhynchus R millefolius R hierosolymitanus R cicutarius R garganicus R millefoliatus R heterorhizus R illyricus R montanus R aduncus R apenninus R pollinensis R sartorianus R marschlinsii R carinthiacus R venetus R pseudomontanus R gouanii R carpaticus R villarsii R afghanicus R aucheri R elbursensis R termei R leptorrhynchus R linearilobus R makaluensis R macropodioides R papyrocarpus R regelianus R paludosus R pseudomillefoliatus R olissiponensis R bullatus R gregarius R spicatus R cortusifolius R gracilis R asiaticus R amblyolobus R buhsei R cappadocicus R breyninus R brachylobus R fascicularis R hispidus R septentrionalis R acriformis R petiolaris R hawaiiensis R mauiensis R maclovianus R orthorhynchus R diffusus R oreophytus R rarae R tenuirostrus R macounii R repens R marginatus R muricatus R cornutus R trilobus R serpens R polyanthemos R sardous R submarginatus R bulbosus R neapolitanus R multifidus R pinnatus R pensylvanicus R silerifolius R cantoniensis R chinensis R caprarum R peduncularis R bonariensis R brutius R dissectus R caucasicus R sojakii R arvensis R arvensis R acris R glabriusculus R japonicus R lanuginosus R granatensis R serbicus R laetus R laetus R taisanensis R grandiflorus R kotschyi R velutinus R baldschuanicus R cassius R occidentalis R uncinatus R stagnalis R tembensis R chius R parviflorus R constantinopolitanus R strigillosus R sericeus R pinardi R cheirophyllus R ficariifolius R volkensii R flagelliformis R ophioglossifolius R lateriflorus R flammula R reptans R lingua R alismifolius R hydrophilus R meyeri IX. Tet hyan cl. K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 R rumelicus R sprunerianus R psilostachys R argyreus R damascenus R oxyspermus R macrorrhynchus R millefolius R hierosolymitanus R cicutarius R heterorhizus R illyricus R garganicus R millefoliatus R montanus R sartorianus R apenninus R marschlinsii R aduncus R venetus R carinthiacus R pseudomontanus R carpaticus R gouanii R pollinensis R villarsii R elbursensis R termei R aucheri R leptorrhynchus R linearilobus R afghanicus R makaluensis R macropodioides R papyrocarpus R regelianus R asiaticus R gracilis R olissiponensis R paludosus R pseudomillefoliatus R spicatus R bullatus R gregarius R cortusifolius R amblyolobus R cappadocicus R buhsei R breyninus R brachylobus R fascicularis R hispidus R acriformis R hawaiiensis R mauiensis R petiolaris R septentrionalis R maclovianus R orthorhynchus R diffusus R tenuirostrus R macounii R repens R cornutus R marginatus R trilobus R caprarum R peduncularis R oreophytus R rarae R serpens R polyanthemos R sardous R submarginatus R bulbosus R neapolitanus R cantoniensis R chinensis R pensylvanicus R silerifolius R muricatus R multifidus R pinnatus R bonariensis R brutius R dissectus R caucasicus R sojakii R arvensis R granatensis R serbicus R laetus R laetus R lanuginosus R taisanensis R acris R glabriusculus R japonicus R baldschuanicus R cassius R kotschyi R velutinus R occidentalis R uncinatus R stagnalis R tembensis R chius R parviflorus R constantinopolitanus R strigillosus R sericeus R pinardii R grandiflorus R cheirophyllus R volkensii R ficariifolius R flagelliformis R ophioglossifolius R lateriflorus R flammula R reptans R lingua R alismifolius R hydrophilus R meyeri BI 10 changes Fig. 1a. Phylogenetic relationships of Ranunculus species and allied genera of the combined plastid and ITS dataset based on Maximum Parsimony analyses (MP), and Bayesian inference (BI). Bootstrap value P50 and posterior probability values P90 are indicated above branches. Tree overview is presented in the upper left-corner. Author's personal copy 7 IV III II I allied genera Ficaria kochii Ficaria verna Coptidium lapponicum Coptidium pallasii Arcteranthis cooleyae Trautvetteria grandis Halerpestes cymbalaria H uniflora Oxygraphis polypetala 10 changes Beckwithia andersonii Cyrtorhynchy ranunculina Peltocalathos baurii Hamadryas delfinii Callianthemoides semiverticillatus Kumlienia hystricula Isopyrum thalictroides Anemone quinquefolia III A, alpine- arctic- wetland clades VI V II VII I VIII allied genera B, meridional-temperate clade IX Out group R pulchellus R brotherusii R pseudopygmaeus R nephelogenes R longicaulis R pseudohirculus R hirtellus R membranaceus R punctatus R adoneus R nivalis R pygmaeus R pedatifidus R cardiophyllus R gelidus R rufosepalus R cassubicifolius R carpaticola R micranthus R notabilis R nipponicus R peltatus R trichophyllus R sphaerospermus R collinus R papulentus R natans R radicans R aquatilis R penicillatus R fuegianus R gmelinii R hyperboreus R sceleratus R lyallii R apiifolius R kuepferi R seguieri R aconitifolius R platanifolius R camissonis R glacialis R cacuminis R bilobus R alpestris R traunfellneri R crenatus R magellensis R amplexicaulis R parnassifolius R acetosellifolius R pyrenaeus R gramineus R brevifolius R hybridus R thora Krapfia clypeata Laccopetalum giganteum Ceratocephala orthoceras Ceratocephala falcata Myosurus minimus IV K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 Fig. 1b. Phylogenetic relationships of Ranunculus species and allied genera of the combined plastid and ITS dataset based on parsimony analyses. Bootstrap value P50 is indicated above branches. Tree overview is presented in the upper left-corner. 1 ll of 0.4% bovine serum albumin (BSA, Promega, Madison, WI, USA), and in the case of the ITS region, dimethyl sulfoxide (DMSO) to reduce problems associated with DNA secondary structure, 1 ll each primer (10 mmol/l) and 1 ll template DNA. PCR products were purified using E. coli Exonuclease I and Calf Intestine Alkaline Phosphate (CIAP; MBI-Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. Cycle sequencing was performed using Big Dye™ Terminator v3.1 Ready Reaction Mix (Applied Biosystems), using the following cycling conditions: 38 cycles of 10 s at 96 °C, 25 s at 50 °C, 4 min at 60 °C. All DNA regions were sequenced in both directions. The samples were run on a 3130xl Genetic Analyzers capillary sequencer (Applied Biosystems). 2.3. Sequence alignment and phylogenetic analysis The sequences were initially aligned using Clustal X (Thompson et al., 1997). Subsequent corrections were carried out manually using BioEdit version 7.0.9.0 (Hall, 1999). Indels were treated as binary characters following the ‘‘simple indel coding method’’ (Simmons and Ochoterena, 2000) using the program SeqState version 1.36 (Müller, 2005), and the data set with gaps coded was used for all analyses. Nuclear and chloroplast sequences were analyzed separately and combined. A heuristic search for most parsimonious (MP) trees was performed with PAUP version 4.0b8 (Swofford, 2002). The analyses involved 1000 replicates with stepwise random taxon addition, tree bisection–reconnection (TBR) and branch swapping saving no more than 10 trees per replicate. All characters were equally weighted and treated as unordered (Fitch, 1971). Strict consensus trees were computed from all equally most parsimonious trees. Internal branch support was estimated using non-parametric bootstrapping (Felsenstein, 1985) with 1000 replicates and 10 addition sequences replicates. To reduce the impact of model misspecification on tree topology accuracy (Sullivan and Swofford, 2001; Nylander, 2004) a model based on Bayesian inference was used to reconstruct phylogeny in addition to maximum parsimony. Different partitions Taxon Country; collector, collection No.; Herbar matK/trnK psbJ–petA GU257978 AY680201 AY680197 AY680199 AY680191 AY680190 AY680194 AY680195 GU257973 AY680193 AY680192 AY680196 GU552270 GU257974 GU257977 GU552271 GU257975 GU552272 AJ347913 GU257976 AY680200 AY680075 AY680081 HQ338296 AY680167 AY680030 AY680088 HQ338297 HQ338298 AY680078 HQ338299 AY680071 AY680091 AY680092 HQ338300 FM242844 AY680177 HQ650550 GU257963 HQ338301 AY680174 AY680077 GU257964 HQ338302 AY680187 AY680115 HQ338303 HQ338304 FM242860 AY680124 AY680114 HQ338305 AY680083 GU257980 – AY954238 AY954236 AY954229 AY954230 AY954234 AY954233 GU257981 AY954231 AY954232 AY954237 GU552273 GU257982 GU257979 DQ490058 GU257983 Peru; Cano & al. 15196; USM; DQ400695 AJ414344 GU257984 AY954235 AY954226 AY954217 HQ338378 AY954199 HM565145 AY954143 HM565146 HM565147 AY954221 HM565148 AY954223 AY954150 AY954140 — FM242780 AY954193 HQ650551 GU257985 HQ338379 AY954195 AY954220 GU257986 HQ338347 AY954212 AY954172 HM565149 HQ338348 FM242796 AY954188 AY954161 HQ338373 AY954218 GU257995 GU258002 GU258003 Gothenberg, BG; Johansson s.n.; GU258004 GU257996 GU257997 GU257997 GU257999 GU258005 GU258000 GU258001 GU258006 Argentina; Weigend 7003; M; GU258007 GU258011 GU258014 – GU258008 Halle, BG; J.T. Johansson s.n.; GU258009 – GU258012 GU258010 HQ338261 HQ338172 HQ338181 GU258015 USA, Utah; Tremetsberger s.n.; WU; HQ338195 HQ338206 HQ338217 HQ338228 HQ338239 HQ338250 HQ338262 HQ338273 Uruguay; Lorentz 533 W; GU258016 HQ338285 HQ338150 HQ650549 GU258017 GU258018 HQ338161 HQ338168 HQ338169 GU258019 HQ338192 GU258020 GU258021 HQ338170 HQ338171 HQ338173 HQ338174 HQ338175 HQ338176 GU258022 Author's personal copy USA – Connecticut; Mehrhoff 12602; CONN Canada; Jensen 28432; MPN Sweden – Gothenburg BG; Johansson s.n.; GB Argentina; Lehnebach s.n.; VALD Iran; Rechinger Jr. 50857; W Austria; Hörandl 3837; WU Sweden; Johansson s.n.; Alaska; Elven & al. SUP02-175; O USA; Nunn 1775; RM Sweden – Gothenburg BG; Johansson s.n.; GB Sweden; Johansson s.n.; – Italy – Rezia BG; Johansson 204; LD Chile; Lehnebach s.n.; MPN Argentina; Schönswetter AR08-20, WU Austria; Hörandl 641; WU Peru; Sanchez & al. 11173 F, CPUN, MPN USA; L. Grant 286 ZT Germany – Halle, BG; J.T. Johansson s.n.; Genbank Nepal; – 1926-3; LI South Africa; Mucina 030103/22; WU Sweden – Gothenburg BG; Johansson s.n.; – Cult. Copenhagen BG; Johansson 274; LD USA, Utah; Albach 844; WU Germany – Bonn BG; Johansson 194; CONN USA, Colorado; Ehrendorfer FER70; WU Italy; Hörandl 6818; WU Iran; Emadzade 114; WU USA; Hörandl 9622; WU Cult. Rezia BG; Johansson 242; LD Iran; Emadzade 120; WU Sweden – Lund BG; Johansson 222; LD Italy; Hörandl 6069; WU Chile; Lehnebach s.n.; VALD USA; Hörandl 9625; WU Turkey; Brause 45; LE Germany – Kiel BG; Johansson 180; CONN Iran; Emadzade 109; WU Iran; Shooshtari 2569; TARI Iran; Emadzade 101; WU Denmark – Copenhagen BG; Johansson 272; LD Italy; Hörandl 4574; WU Argentina; Schönswetter AR08-2a; WU Iran; Emadzade 115; WU Sweden – Gothenburg BG; Johansson s.n;. GB Austria; Hörandl 5249; WU Nepal; Hörandl & Emadzade 9678; WU Italy; Pittoni s.n.; M Russia; Ahrns –; HAL Sweden; Johansson s.n.; – Greece; Hörandl & Gutermann 7191; WU Greece; Huberk & Krug 13565; Z & ZT USSR; Koropewa s.n.; W GenBank accession Nos. ITS K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 Anemone quinquefolia L. Arcteranthis cooleyae (Vasey & Rose) Greene Beckwithia andersonii (A. Gray) Jeps. Callianthemoides semiverticillatus (Philippi) Tamura Ceratocephala falcata (L.) Pers. C. orthoceras DC. Coptidium lapponicum (L.) Tzvelev C. pallasii (Schlecht.) Tzvelev Cyrthorhyncha ranunculina Nutt ex. Torr. & A. Gray. Ficaria fascicularis K.Koch F. verna Huds. ssp. verna Halerpestes cymbalaria (Pursh) Greene H. uniflora (Phil. ex. Reiche) Emadzade et al. Hamadryas delfinii Phil. Isopyrum thalictroides L. Krapfia clypeata (Ulbr.) Standl. & J.F.Macbr. Kumlienia hystricula (A.Gray) E. Greene Laccopetalum giganteum Ulbr. Myosurus minimus L. Oxygraphis polypetala Hook. F. & Thomson. Peltocalathos baurii (McOwan) Tamura R. acetosellifolius Boiss. R. aconitifolius L. R. acriformis A. Gray R. acris L. R. adoneus A. Gray R. aduncus Gren. & Godr. R. afghanicus Aitch. & Hemsl. R. alismifolius Geyer ex Benth. R. alpestris L. R. amblyolobus Boiss. & Hohen. R. amplexicaulis L. R. apenninus (Chiov.) Pign. R. apiifolius Pers. (Aphanostemma apiifolia) R. aquatilis L. R. argyreus Boiss. R. arvensis L. R. arvensis L. R. asiaticus L. R. aucheri Boiss. R. baldschuanicus Regel ex Kom. R. bilobus Bertol. R. bonariensis Poir. R. brachylobus Boiss. & Hohen. R. brevifolius ssp. brevifolius Ten. R. breyninus Cr. R. brotherusii Freyn R. brutius Tenore R. buhsei Boiss. R. bulbosus ssp. bulbosus L. R. bullatus L. R. cacuminis Strid & Papan. R. camissonis Aucl. 8 Table 1 Materials, voucher information and GenBank accession numbers. Taiwan; Huang 1975; HAST Georgia, Caucasus; Hörandl 8269; WU Chile; Juan Fernandez Isl., Landero 9355, OS Sweden – Gothenburg BG; Johansson HZ 86-29, GB Austria; Hörandl 4096; WU Slovakia; Hörandl 8483; WU Romania; Paun s.n.; WU Lebanon; Maitland 289; LE Germany; Hörandl 8476; WU Georgia; Hörandl 8259; WU Taiwan; Hörandl 9550; WU Russia; Khakevich & Buch 1355; ZT Greece; Gutermann & al. 34758; WU Iran; Akhani 320156; Li Canberra BG; Crisp & Telford 2227; CAN Iran; Memarianii 117; WU Azerbaijan; Schneeweiss 6806; WU Cult. Halle BG; Johansson 237;LD Austria; Hörandl 2818; WU Turkey; Nydegger 41126; ZT Nepal; Hörandl & Emadzade 9706; WU Turkey; Walther 9258; LE Iran; Emadzade 105; WU USA Pennsylvania; Keener 2004-1; WU Peru; Gute & Müler 309853; Li Germany – Oldenburg BG; Johansson 193; CONN Nepal; Hörandl & Emadzade 9677b; WU Chile; L. & F. Ehrendorfer s.n.; VALD Greece; Gutermann & al. 34974; WU Xinjiang, China; Wang 28426; MPN Russia; Skvortsov 10913; M Sweden; Johansson s.n. – USA, Alaska; Schröck 454907; LI Cult. Schachen; Johansson s.n. – Greece; Johansson s.n. – cult. Krefeld BG; Johansson s.n. – unknown; Johansson 266; LD Georgia; Hörandl 8271; WU Cult. Berlin – Dahlem BG; Johansson 232; LD USA; Jeffery 650079; BISH Turkey;Nydegger; 46083; M Palestina; Favrat s.n.; ZT Nepal; Tod 372997; LI USA, Pennsylvania; Keener 2004-3b; WU Sweden, Gothenburg BG; Johansson s.n. GB Argentina; Schönswetter AR08-10; WU Sweden; Johansson s.n. – Sweden; Lundgren s.n.; – China; XieLei XL200348; WU Iran; Emadzade 113; WU Austria; Hörandl 4336; WU India; Lone 1750; WU India; Lone 1761; WU Unknown; Johansson 255; LD Cult. Catania BG; Johansson 235; LD Iran; Emadzade 111; WU Afghanistan; Podlech 10374; M Cult.Lund BG; Johansson s.n.; – Pakistan; Millinger 470564; LI HQ338306 AY680117 AY680151 AY680045 AY680093 AY680041 AY680096 FM242848 AY680040 AY680178 GU257965 HQ338307 AY680176 AY680103 AY680059 HQ338308 AY680153 AY680101 AY680086 HQ338309 HQ338310 FM242849 HQ338311 HQ338312 AY680182 AY680185 HQ338313 AY680064 AY680107 AY680054 HQ338315 AY680082 AY680063 AY680098 AY680120 AY680076 AY680165 AY680053 AY680100 HQ338316 HQ338317 HQ338318 AY680038 HQ338319 AY680189 – AY680065 AY680119 AY680164 HQ338320 AY680085 HQ338321 HQ338321 AY680163 AY680179 HQ338323 HQ338324 AY680184 AY680051 HM565150 AY954173 HM565151 AY954124 AY954145 AY954111 AY954154 FM242784 AY954112 AY954192 GU257987 HQ338349 AY954201 AY954167 AY954137 HQ338350 AY954178 AY954160 AY954228 HM565153 HQ338351 FM242785 HQ338352 HM565154 AY954208 AY954204 HQ338375 AY954136 AY954165 AY954114 HQ338353 AY954219 AY954128 AY954151 AY954171 AY954227 AY954197 AY954203 AY954159 HM565155 HM565156 HQ338354 AY954120 HQ338355 AY954211 HM565157 AY954135 AY954162 AY954200 HQ338356 AY954213 HM56515 HQ338357 AY954194 AY954209 HQ338358 HQ338359 AY954206 AY954117 HQ338177 HQ338178 HQ338193 HQ338179 HQ338180 FJ619866 HQ338182 HQ338183 FJ619867 GU258023 GU258024 HQ338184 HQ338185 HQ338186 HQ338187 HQ338188 HQ338189 Portugal, Madeira; Hörandl 9586, WU; HQ338190 HQ338191 HQ338196 HQ338197 HQ338198 HQ338199 HQ338201 HQ338202 GU258025 HQ338203 Argentina; Schönswetter Ar08-14; WU; HQ338194 HQ338204 HQ338205 HQ338207 GU258027 FJ619879 HQ338209 HQ338210 HQ338211 HQ338212 HQ338213 HQ338214 HQ338215 HQ338216 HQ338218 HQ338220 HQ338221 HQ338222 HQ338223 HQ338224 HQ338225 HQ338226 HQ338227 GU258028 HQ338229 HQ338230 HQ338231 HQ338233 HQ338234 HQ338235 HQ338236 HQ338237 9 (continued on next page) Author's personal copy cantoniensis DC. cappadocicus Willd. caprarum Skottsb. cardiophyllus Hook. carinthiacus Hoppe carpaticola Soó carpaticus Herbich cassius Boiss. cassubicifolius W. Koch caucasicus MB. cheirophyllus Hayata chinensis Bunge chius DC. cicutarius Schlecht. collinus DC. constantinopolitanus (DC.) d’Urv. cornutus DC. cortusifolius Willd. crenatus Waldst. & Kit. damascenus Boiss. & Gaill. diffusus DC. dissectus M. Bieb. var. napellifolius (DC.) P.H. Davis elbursensis Boiss. fascicularis Muhl. flagelliformis Sm. flammula L. ficariifolius Leveill & Van fuegianus Speg. garganicus Ten. gelidus Kar. & Kir. glabriusculus Rupr. glacialis L. gmelinii ssp. gmelinii DC. gouanii Willd. gracilis Schleich. gramineus L. granatensis Boiss. grandiflorus L. gregarius Brot. hawaiiensis A. Gray heterorhizus Boiss. & Bal. hierosolymitanus Boiss. hirtellus Royle hispidus Michx. hybridus Biria hydrophilus Gaudich. hyperboreus Rottb. illyricus L. japonicus Thunb. kotschyi Boiss. kuepferi Greuter & Burdet laetus Wallich ex D.Don laetus Wallich ex D.Don lanuginosus L. lateriflorus DC. leptorrhynchus Aitch. & Hemsl. linearilobus Bunge lingua L. longicaulis C.A.Mey. K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. 10 Table 1 (continued) Taxon New Zealand; Steel 24603; MPN Chile;.Lehnebach s.n.; VALD Canada; Alsos & Brysting CA72; ? Iran; Mozaffarian 77929; TARI Iran; Emadzade 108; WU Italy; Baltisberger & Krug 12831; Z & ZT Nepal; Hörandl & Emadzade 9700; WU Cult. Copenhagen BG; Johansson 286; LD Corse; Hörandl 6981; WU USA; Oppenheimer 684216; WU Nepal; Hörandl & Emadzade 9696; WU South Africa; Gehrke et al. BG-Af 463, ZH USA, Ohio; Lonsing 50563; Li Cult. Graz BG; Johansson 293; LD Iran; Emadzade 131; WU Austria; Hörandl 666; WU South Africa; Mucina 031102/7; WU Cult. Siena BG; Johansson 210; LD Russia; Tribsch 9558; WU Greece; Johansson 224; LD Pamir; Dickore 17912; M Russia; Egorova s.n.; LE Sweden; Johansson s.n.; –. Austria; Hörandl 5612; WU USA; Pykälä & Norris, 1139 W Spain; Gutermann 37407; WU France – Nantes BG J.T. Johansson 208 LD Ethiopia; Gehrke et al. BG-Af 209, ZH USA, Hörandl 9618 WU, UT Iran; Emadzade 100; WU Greece; Gutermann & al. 34754; WU Cult. Canberra BG;Johansson 760141p; – Iran; Tajeddini 110; WU France/Spain Schneeweiss & al. 6509; WU cult. Copenhagen BG; Johansson 287; LD USA; Orthner 593; RM Chile; Lehnebach s.n.; VALD France – Nantes BG; Johansson 206; LD England; G. Dahlgren BE9; LD USA; Zila 447002; LI Mexico; Stuessy 18581; WU Iran; Ghahremanii 108; WU Madagascar; Gehrke et al. BG-Af 247, ZH Norway; Johansson 277; LD Italy; Hörandl 8247; WU Austria; Hörandl 5130; WU Russia; Tribsch 9593; WU Spain; Schneeweiss & al. 7253; WU Slovakia; Hörandl 5904; WU Nepal; Hörandl & Emadzade 9689; WU Sweden – Lund BG; Johansson 219; LD Nepal; Hörandl & Emadzade 9679; WU Russia; Zimarskaya & al. s.n., LE Sweden; Larson & Granberg 9345; WU matK/trnK psbJ–petA AF323277 AY680158 HQ338325 HQ338326 HQ338327 HQ338328 HQ338329 AY680150 AY680089 HQ338330 HQ338331 EU288400 AY680042 AY680108 HQ338332 AY680094 AY680162 AY680148 AY680113 AY680123 HQ338333 FM242834 AY680046 AY680033 HQ338334 AY680109 AY680180 EU288412 HQ338335 GU257967 AY680102 AY680058 GU257968 AY680072 AY680175 GU257969 AY680154 AY680068 AY680070 AY680147 HQ338336 GU257970 EU288415 AY680080 AY680097 AY680121 AY680111 AY680110 AY680090 HQ338314 AY680106 HQ338337 FM242818 AY954242 AY954142 AY954181 HM565159 HQ338360 HM565160 HQ338376 HM565161 AY954177 AY954147 HM565162 HQ338361 EU288374 AY954113 AY954166 HQ338362 AY954149 AY954183 AY954191 AY954134 AY954187 HQ338363 FM242770 AY954123 AY954115 HM565164 AY954157 AY954207 HQ338377 HQ338364 GU257989 AY954155 AY954138 GU257990 AY954224 AY954202 GU257991 AY954180 AY954131 AY954130 AY954190 HQ338365 GU257992 EU288388 AY954216 AY954152 AY954185 AY954118 AY954156 AY954146 HQ338374 AY954170 HM565165 FM242754 AY954122 New Zealand; Schneeweiss & al. WU, GU258029 Argentina; Schönswetter AR08-17 WU; GU258030 HQ338238 HQ338240 HQ338241 HQ338242 HQ338243 HQ338244 HQ338245 HQ338246 HQ338247 HQ338248 HQ338249 HQ338251 HQ338252 HQ338253 HQ338254 HQ338255 GU258031 HQ338256 HQ338258 HQ338259 GU258032 FJ619873 HQ338263 HQ338264 HQ338265 HQ338266 HQ338267 GU258033 HQ338268 HQ338269 GU258034 GU258035 HQ338270 GU258036 – GU258037 HQ338272 GU258038 HQ338274 GU258039 HQ338275 HQ338276 HQ338277 GU258040 HQ338278 HQ338279 HQ338280 HQ338219 HQ338281 HQ338282 HQ338284 HQ338232 Author's personal copy lyallii Hook. f. maclovianus Urv. macounii Britton macropodioides Briq. macrorrhynchus Boiss. magellensis Ten. makaluensis Kadota marginatus Urv. marschlinsii Steud. mauiensis A. Gray membranaceus Royle meyeri Harv. micranthus Nutt. millefoliatus Vahl millefolius Banks & Soland. montanus Willd. multifidus Forssk. muricatus L. natans C.A.Mey. neapolitanus Ten. nephelogenes Edgew. nipponicus Nakai nivalis L. notabilis Hörandl & Guterm. occidentalis Nutt. olissiponensis Pers. ophioglossifolius Vill. oreophytus Delile orthorhynchus Hook. oxyspermus Willd. paludosus Poir. papulentus Melville papyrocarpus Rech. F., Aell. & Esfand. parnassifolius ssp. parnassifolius L. parviflorus L. pedatifidus J.E. Smith, peduncularis Sm. peltatus ssp. peltatus Moench penicillatus ssp. pseudofluitans (Dum.) Bab. pensylvanicus L. petiolaris Kunth ex DC. pinardii (Stev.) Boiss. pinnatus Poir. platanifolius L. pollinensis Chiovenda polyanthemos L. pseudohirculus Schrenk ex F.E. Fischer & C.A. Mey. pseudomillefoliatus Grau pseudomontanus Schur cf. pseudopygmaeus Hand.-Mazz. psilostachys Griseb. pulchellus C.A.Mey punctatus Jurtzev pygmaeus Wahlenb. GenBank accession Nos. ITS K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. R. Country; collector, collection No.; Herbar AY680074 FM242857 EU288416 HQ338338 HQ338339 AY680186 AY680047 AY680104 AY680122 AY680095 GU257971 AY680079 FM242832 AY680166 HQ338340 AY954243 HQ338341 HQ338342 AY680066 AY954244 AY680105 EU288419 HQ338343 FM242841 HQ338344 EU288421 HQ338345 HQ338346 AY680188 AY954245 AY680067 AY680149 GU257972 AY680173 AY680087 AY680099 EU288424 AY680202 AY954225 FM242793 EU288389 HQ338366 HM565166 AY954205 AY954121 AY954168 AY954186 AY954148 GU257993 AY954215 FM242768 AY954196 HM565167 AY954184 HQ338367 HQ338368 AY954132 AY954158 AY954169 EU288392 HQ338369 FM242777 HQ338370 EU288393 HQ338371 HQ338372 AY954210 AY954222 AY954133 AY954176 GU257994 AY954198 AY954144 AY954153 EU288396 AF007945 GU258041 HQ338260 HQ338286 HQ338271 HQ338287 HQ338288 GU258042 HQ338289 HQ338290 HQ338283 GU258043 HQ338291 HQ338292 HQ338293 HQ338294 HQ338257 HQ338295 HQ338151 GU258044 HQ338152 HQ338153 HQ338154 HQ338155 HQ338156 HQ338157 HQ338158 HQ338159 HQ338160 GU258045 HQ338162 GU258046 HQ338163 GU258047 HQ338164 HQ338165 HQ338166 HQ338167 GU258013 Author's personal copy Spain; Schneeweiss & al.; 6498 WU Mongolia; Schamsran 44272, HAL Malawit; Gehrke et al. BG-Af 304, ZH Pamir, Vasak s.n.; W Iran; Emadzade 107; WU Switzerland; Willi br3; Z Pakistan; Millinger392897; Li Greece; Snogerup 5993b; LD Sweden; Johansson s.n.; – Cult. Copenhagen BG; Johansson 271; LD Iran; Emadzade 112; WU Sweden – Gothenburg BG; Johansson 226; LD USA; Raven et al. 27447; LE Cult. Mühlhausen BG; Johansson 249; LD Iran; Emadzade 121; WU Austria; Hörandl 9522; WU Taiwan; Huang 1884; HAST Iran; Emadzade 122; WU Turkey; Dahlgren B87B; LD cult. Wisley Bot. Garden, Johansson s.n. LD Greece; Johansson 230; LD Ethiopia; Gehrke & al. BG-Af 228; ZH Iran; Emadzade 117; WU Russia, Altai; Pobedimova 52; LE Taiwan; Yang & al. 7474; TNM Ethiopia; Gehrke & al. BG-Af 210; ZH China; Podlech 55472; M Iran; Mozaffarian 54814; TARI Sweden – Lund BG; Johansson 223; LD Austria; Hörandl 2518; WU Greece; DahlgrenB23; LD Belgium – Antwerpen BG; Johansson 217; LD. USA; Holmgren 5379; ZT The Netherlands – Rotterdam BG; Johansson 270; LD Italy; Gutermann & al. 35349; WU Austria; Hörandl 664; WU Uganda; Gehrke & al. BG-Af 353; ZH USA – California BG; Johansson 82.1322; – K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 R. pyrenaeus L. R. radicans C.A. Mey. R. rarae Exell R. regelianus Ovcz. R. repens L. R. reptans L. R. rufosepalus Franch. R. rumelicus Griseb. R. sardous Cr. R. sartorianus Boiss. & Heldr. R. sceleratus L. R. seguieri ssp. seguieri Vill. R. septentrionalis Poir. R. serbicus Vis. R. sericeus Banks & Soland. R. serpens ssp. nemorosus (DC.) G. Lopez Gonzalez R. silerifolius Lev. R. sojakii Iranshahr & Rech. f. R. sphaerospermus Boiss. & Blanche R. spicatus Desf. R. sprunerianus Boiss. R. stagnalis Hochst. ex A. Rich. R. strigillosus Boiss. & Hutt R. submarginatus Ovcz. R. taisanensis Hayata R. tembensis Hochst. ex A. Rich. R. tenuirostris J.Q.Fu R. termei Iranshahr & Rech. f. R. thora L. R. traunfellneri Hoppe R. trichophyllus Chaix R. trilobus Desf. R. uncinatus D. Don. R. velutinus Schur R. venetus Huter ex Landolt R. villarsii DC. R. volkensii Engl. Trautvetteria grandis Honda 11 Author's personal copy 12 K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 of the data set, ITS, matK, trnK, and psbJ–petA, were tested using Mr. Modeltest 2.2 (Nylander, 2004) separately to determine the sequence evolution model that best described the present data. The GTR + I + C substitution model was selected as the best fitting model by both a hierarchical likelihood ratio test and Akaike information criterion as implemented in MrModeltest version 2.2 (Nylander, 2004) for all partitions, and it was used for final analysis using Mr. Bayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Four Markov chains were run simultaneously for 5000,000 generations, and these were sampled every 1000 generations. After the chains had reached stationarity as judged from plots of likelihood and from split variances being <0.01, data from the first 10% of generations were discarded as the ’’burn-in’’. A majority rule consensus tree was constructed and posterior probabilities (PP) of nodes were calculated from the remaining sample. 2.4. Optimization of ancestral distributions For biogeographical investigations we chose the well-resolved meridional-temperate clades V–IX (Fig. 1a, flammula, acris, polyanthemos, arvensis, and Tethyan clades) from the analyses of the complete data set (see above), and analyzed each of them separately because of too low resolution in the backbone of the tree topology. We used the parsimony based methods as implemented in DIVA (DIVA v. 1.1, Ronquist, 1997) and Mesquite (Mesquite v. 2.6, Maddison and Maddison, 2009) and also the maximum likelihood method implemented in Lagrange (Lagrange v. 2.0.1, Ree and Smith, 2008) to infer vicariance and dispersal events. DIVA assumes that speciation is the result of vicariance, e.g. either a split of a wide distribution in two areas, or a speciation event within a single area, in which the two daughter species remain in their native area immediately following speciation (Ronquist, 1996, 1997). A newly developed method represents a significant advance in biogeographic methodology by using a maximum likelihood (ML) statistical model (Lagrange; Ree and Smith, 2008). The program Lagrange not only finds the most likely ancestral areas at a node and the split of the areas in the two descendant lineages, it also calculates the probabilities of these most-likely areas at each node (Ree and Smith, 2008). This method includes information from biological and biotic factors by calculating the likelihood of biogeographical routes and areas occupied by most common ancestor for a given phylogenetic tree topology and the present distributions of taxa. For example, the rate of dispersal and local extinction, the time of lineage survival, and the probabilities of dispersal between geographic ranges at different geological times (Ree et al., 2005) can all be incorporated in the reconstruction. Lagrange was employed here with a simple model of one rate of dispersal and extinction constant over time and among lineages (Emadzade and Hörandl, in press). We further reconstructed ancestral states based on Fitch parsimony optimization (FPO) using Mesquite (Maddison and Maddison, 2009). Fitch parsimony calculates the most parsimonious ancestral states at the nodes of the tree assuming free reversibility for multistate characters and one step per state change. In general, the FPO allows for fewer possible ancestral area reconstructions than DIVA, because only terminals can have more than one state (i.e., areas). Therefore, FPO assumes that geographic distributions are the result of dispersal events rather than vicariance (e.g., Kron and Luteyn, 2005). To run Lagrange, an ultramatric tree is needed so we used ultrametric trees using the Bayesian analysis program BEAST v1.4.5 (Drummond and Rambaut, 2007) for all biogeographical analyses. The partitioned BEAST.xml input file was created with BEAUti v1.4.5 (Drummond and Rambaut, 2007). A GTR + I + C substitution model which was determined as the best model for our data set using Mr. Modeltest 2.2 (Nylander, 2004) and the gamma distribution were modeled with four categories. A Yule prior on branching rates was employed and four independent MCMC analyses were each run for 10,000,000 generations, sampling every 1000 generations. After discarding the first 10% of generations of each run as burn-in, the four runs were combined using TreeAnnotator (Rambaut and Drummond, 2002) to obtain an ultramatric tree. Distribution data were compiled from the literature (e.g., Ovczinnikov, 1937; Meusel et al., 1965; Lourteig, 1984; Riedl and Nasir, 1990; Iranshahr et al., 1992; Tutin and Cook, 1993; Rau, 1993; Whittemore, 1997; Wencai and Gilbert, 2001) to assign species to the five major geographic areas: Eurasia, N. America, S. America, Africa, and Hawaii. Mediterranean species that extend their distribution along the coast of North Africa were coded as Eurasian only. The distribution of each species of Ranunculus included in this analysis is shown in Figs. 2a and 2b, and Appendix 1. For the study of details of historical biogeography in the Eurasian clades (‘‘Tethyan clade’’) as a model of a restricted area, Eurasia was subdivided into an eastern and a western Mediterranean area (including North Africa), Circumboreal, Irano-Turanian (excluding C. Asian high mountains), Central Asia, Eastern Asia, and the Himalaya-India region. Because the distribution matrix containing 48 taxa and six areas was too large to be read by DIVA, the tree was subdivided into two parts (Fig. 2b, sections A and B). The optimization was performed in two steps: first, analysis of section A was processed alone and reduced to a single branch with its optimized areas; second, analysis of section B included this single branch (Ronquist, 1996). 3. Results 3.1. Phylogenetic analyses The MP analyses of all 205 species based on ITS revealed 70 most parsimonious trees with CI = 0.3609 and RI = 0.8241, while chloroplast markers only (matK/trnK, psbJ–petA) revealed 60 most parsimonious trees with CI = 0.4772 and RI = 0.8503. The MP analysis of combined data resulted in 1120 most parsimonious trees with CI = 0.4152 and RI = 0.8260. The ITS analysis did not resolve well relationships within Ranunculus, showing a basal polytomy (not shown). The strict consensus tree of the chloroplast DNA overall showed better statistical support. As in previous studies (Paun et al., 2005; Gehrke and Linder, 2009; Hoffmann et al., 2010), the main clades were retained with both datasets (not shown). Parsimony analysis of the combined data set revealed a better resolution and higher statistical support than the results of either data set alone (Figs. 1a and 1b). The topology provided by maximum parsimony (MP) of the combined data displays nine well-supported clades (Figs. 1a and 1b; clades I–IX) which represent widespread ecological groups as in previous studies (Johansson, 1998; Hörandl et al., 2005; Paun et al., 2005; Lehnebach, 2008; Gehrke and Linder, 2009; Hoffmann et al., 2010). The monophyly of these clades is well supported; however, their relationship between each other yields only weak support. Bayesian inference (BI) reveals the same clades (I–IX) as the MP tree topology. In general the majority rule consensus tree from the Bayesian analysis showed overall higher resolution between clades and high posterior probabilities (PP) for clades (Fig. 1a). Both analyses confirm the separation of allied genera from Ranunculus s.str. (Fig. 1b). The core Ranunculus clade shows a gross subdivision into a group of clades, tending to colder and more humid areas, or aquatic habitats (Fig. 1b, clade A, I–IV), while a big clade with 94% BS and 100% PP units most species from the (boreal) temperate to meridional (tropical) zones, tending to mesic and dry habitats (Fig. 1a, clade B, V–IX). Author's personal copy K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 13 Fig. 2a. Biogeographical optimization performed with the software DIVA, Lagrange, and Mesquite of meridional-temperate clade of Ranunculus: polyanthemos clade; acris clade; flammula clade. This tree is based on the combined plastid and ITS dataset. Relevant nodes are numbered (in circles). Distributions of MRCAs reconstructed by DIVA are indicated on each node. Age estimates for main nodes are indicated in My (Emadzade and Hörandl, in press), 95% high posterior density are given in parentheses. Different line signatures show the highest probability migration routes suggested by Lagrange. Coloring shows ancestral area reconstruction under parsimony in Mesquite. Nodes of interest for this study are indicated by bold margin. Coded as stated in the figure: N, N. America; S, S. America; E, Eurasia; F, Africa; H, Hawaii. Author's personal copy 14 K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 Fig. 2b. Biogeographical optimization performed with the software DIVA, Lagrange, and Mesquite of meridional-temperate clade of Ranunculus: Tethyan clade; arvensis clade. This tree is based on the combined plastid and ITS dataset. Relevant nodes are numbered and indicated by bold margin (in circles). Distributions of MRCAs reconstructed by DIVA are indicated on each node. Age estimates for main nodes are indicated in My (Emadzade and Hörandl, in press). Different line signatures show the highest probability migration routes suggested by Lagrange. Coloring shows ancestral area reconstruction under parsimony in Mesquite. Sections A and B refer to splitting of the tree for DIVA analysis (see materials and methods). Coded as stated in the figure: C, Circumboreal; W, W. Mediterranean and Macaronesia; E, E. Mediterranean; I, IranoTuranian; A, C. Asia; H, Himalaya and India. Clades I–IV (Fig. 1b, clade A, alpine-arctic-wetland clades) mainly comprise species of high altitudes, latitudes, and wetlands. These clades show in general a low resolution which may be due to reticulate evolution, polyploidy and/or rapid speciation (see Hörandl et al., 2005). The flammula clade (Fig. 1a, clade V) has a basal position in clade B with 100% BS and PP. The Tethyan, arvensis, acris, polyanthemos clades formed a tetrachotomy (Huber, 2003) in either MP or BI analyses with 100% BS and PP (Fig. 1a), although each of these clades is well supported. The arvensis clade and the Tethyan clade (Fig. 1a, clades VI, IX) consist of Eurasian species, and both are well supported in both MP and BI analyses (100% BS, 100% PP). The two remaining well-supported clades (acris, polyanthemos clades; 100%, 91% BS, respectively, and 100% PP for both) comprise species from all continents: Eurasia, North America, South America, Africa, and Oceania. 3.2. Biogeographical analyses Ancestral area reconstructions from DIVA and Lagrange analyses on clades V–IX resulted in more or less similar distribution ranges for most of the nodes (Appendix 1). The most recent common ancestor (MRCA) of the flammula clade (Fig. 1a, clade V) occurred in four areas according to DIVA: Eurasia, Africa, North America, and South America (Fig. 2a, node (1). Ranunculus meyeri from Africa and Ranunculus hydrophilus from South America are sister in this clade (Fig. 2a, node 2, 53% BS, 100% PP). DIVA reconstructed the MRCA of these two species in both Africa and South America, although, Lagrange and Mesquite revealed the MRCA only in Africa, and equivocal, respectively. South American species (Ranunculus flagelliformis) and Eurasian species (Ranunculus ophioglossifolius) formed a clade with 100% PP (Fig. 2a, node 3). Author's personal copy K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 Lagrange and Mesquite suggested that the current distribution of these two species is due to dispersal from Eurasia to South America (with 0.9166 Rel. Prob., in Lagrange). DIVA, however, suggested a vicariance event for this node. The African species Ranunculus volkensii shows close relationships to eastern Asian and Indian species (Fig. 2a, node 4, 69% BS, 100% PP). Lagrange and Mesquite revealed that Eurasia was occupied by the MRCA of this clade (with 0.7199 Rel. Prob., in Lagrange). DIVA however, placed the MRCA within Eurasia and Africa and suggested vicariance for the current distribution of these species. The acris clade (Fig. 2a, clade VI) comprised Eurasian, North American, and African species. DIVA, Lagrange and Mesquite analyses reconstructed Eurasia as the ancestral area of this clade (Fig. 2a, node 5). Some North American species (Ranunculus occidentalis and Ranunculus uncinatus) are sister to a Eurasian clade (Ranunculus baldschuanicus and Ranunculus cassius), which is sister to an African clade (Ranunculus stagnalis and Ranunculus tembensis), but without high support (Fig. 2a, node 6). Lagrange and Mesquite reveals the MRCA of this clade in Eurasia (with 0.7918 Rel. Prob., in Lagrange). DIVA reconstructed the distribution of the MRCA in all three areas or in Eurasia and Africa (Fig. 2a, node 6). The polyanthemos clade (Fig. 2a, clade VIII) is widespread. DIVA reconstructed the ancestral area of this clade in Eurasia, North America, Africa, and South America or in Eurasia and South America (Fig. 2a, node 7). Biogeographical analyses revealed multiple colonizations of all regions. There is evidence of disjunctions of North American and Eastern Asian species (Ranunculus pensylvanicus, Ranunculus silerifolius, Fig. 2a, node 8, 82% BS and 100% PP) and of North American and Eurasia (Ranunculus macounii and Ranunculus repens Fig. 2a, node 9, 96% BS and 100% PP) in this clade. In both cases, DIVA reconstructs the MRCA of these species in Eurasia and North America but Lagrange and Mesquite revealed it in Eurasia (with 0.6628 and 0.6712 Rel. Prob. respectively, in Lagrange). Africa has been colonized at least two times independently in this clade which confirmed previous studies (Gehrke and Linder, 2009). The colonization of the Hawaiian archipelago occurred from North America (Fig. 2a, node 11). The arvensis clade (Fig. 2b, clade VII) includes five species. The Eurasian widespread Ranunculus arvensis is sister to the remaining species which have restricted distribution areas in the western Irano-Turanian and eastern Mediterranean regions. DIVA and Lagrange analyses showed that the MRCA of this clade had a widespread distribution in Eurasia identical to the area currently inhabited by Ranunculus arvensis (Fig. 2b, node 12), while the Mesquite analyses reconstructed the distribution of the MRCA in the Irano-Turanian region. None of the biogeographical analyses revealed the ancestral area of the Tethyan clade (Fig. 2b, clade IX) unambiguously. The biogeographical history in the Tethyan clade shows several colonization events of the Irano-Turanian, the western and the eastern Mediterranean regions. Eastern Mediterranean species mostly show close relationships to Irano-Turanian species, rather than to western Mediterranean and Macaronesian species. Circumboreal species and high mountain European species are nested within the Tethyan clade, indicating a migration of buttercups from lower to higher altitudes/latitudes (Fig. 2b, nodes 20 and 21). 4. Discussion 4.1. Spatial–temporal development of the temperate–meridional clade buttercups The phylogenetic analysis of temperate–meridional groups revealed that closely related species occupy very distinct ranges with intercontinental disjunctions in clades V, VI, and VIII (Fig. 2a), as it 15 was observed in related genera as well (Emadzade and Hörandl, in press). However, the processes that have caused the frequent disjunctions between continents can be only understood in a global spatial–temporal framework. Previous age estimates showed that the stem and crown age group of the temperate–meridional clade (B) date back to the early and mid Miocene, respectively (Paun et al., 2005; Hoffmann et al., 2010; Emadzade and Hörandl, in press). However, the dating approach of Emadzade and Hörandl (in press) provided the highest accuracy for terminal nodes, because it used one external and three internal calibrations, while the earlier studies had just one external calibration for the whole tribe. Therefore, we used the age information of Emadzade and Hörandl (in press) for dating the main biogeographical events (Fig. 2a and 2b). We emphasize on the results of Lagrange analyses because of the limitations of DIVA to reconstruct ancestral areas correctly when speciation has been complicated and has been driven not only by vicariance (Kodandaramaiah, 2010). We focus our conclusions on well-supported nodes that are congruent in all phylogenetic reconstructions. Since the sampling in clades V–VIII covers the meridional to temperate zones for all continents, it is appropriate for understanding intercontinental disjunctions. Clade IX indicates that a more comprehensive sampling adds mainly information on migrations and radiations within continents, but does not influence so much the geographical patterns between continents. The Eurasian arvensis and the Tethyan clades show distinct geographical groups, suggesting regional radiations after geographical separation. We use the well-sampled Tethyan clade for a reconstruction of migration and diversification processes in Eurasia that might have occurred in other continents as well. 4.2. Inter and intracontinental disjunctions 4.2.1. North American–Eurasian disjunctions At least one disjunction event between East Asia (R. silerifolius) and North America (R. pensylvanicus) has happened in the Pleistocene (Fig. 2a, node 8, 82% BS, 100% PP), perhaps as a result of migrations from East Asia to North America (suggested by Mesquite and Lagrange; only DIVA suggested a vicariance event). This migration could have happened across the Bering Land Bridge (BLB) or via long-distance dispersal (LDD) across the Pacific Ocean about 1–2 million years ago (Ma) (Fig. 3, arrow 7). The BLB has been thought to be a region of intercontinental exchange, and was believed to be available through most of the Cenozoic (Tiffney and Manchester, 2001). There is evidence that Beringia remained ice-free during the full glacial events of the Pleistocene and existed as a refugial area (Hulten, 1937; Hopkins, 1959; Yurtsev, 1974; Cook et al., 2005). A connection of Eastern Asian and North America via Oceania and Australia is another reasonable hypothesis. Previous phylogenetic studies placed R. pensylvanicus as sister to a big clade comprising species from the Malesian mountains and from Australia (Hörandl et al., 2005; Lehnebach, 2008). Since these studies did not include Eastern Asian species and were based on ITS sequence data only, they did not provide a robust phylogenetic framework for a biogeographical hypothesis. However, even if Oceania were involved in migration routes, transoceanic dispersal between areas must have occurred as well. The presence of other highly supported disjunctions (96%, 100% PP) in the polyanthemos clade (Fig. 2a, node 9; R. macounii/R. repens) supports an exchange between North America and Eurasia. There are three possibilities to explain this distribution pattern: first, one could assume the presence of a widespread common ancestor in both areas split up by vicariance after the break-up of the North Atlantic Land Bridge (NALB) or Beringia Land Bridge (BLB), followed by allopatric speciation, as suggested by DIVA. Author's personal copy 16 K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 7 9 3 11 9 10 4 2 Fig. 3. Intercontinental biogeographical connections at the tips of the phylogeny of meridional–temperate clade of Ranunculus. Arrows show important dispersal events between continents. Numbers in circles referred to the nodes of the tree in Fig. 2a. Black circles represent terminal taxa. Second, dispersal could have occurred from Eurasia to North America aided by a number of stepping stones; third, a single long-distance dispersal event could explain the observed pattern of distribution. Both Lagrange and Mesquite support a dispersal hypothesis. A vicariance scenario has been observed by a number of studies that have investigated the southwest North AmericanMediterranean disjunctions (Fritsch, 1996, 2001; Liston, 1997; Hileman et al., 2001; Davis et al., 2002). The migration of plants by hopping across the island chains is considered possible mainly in the Miocene (Wen, 1999; Tiffney, 2000; Manos and Donoghue, 2001). Because of the age of this split (Pleistocene, Fig. 2a, node 9; Emadzade and Hörandl, in press), a migration via the BLB is most likely. This pattern has also been reported in related genera of Ranunculeae (Emadzade and Hörandl, in press) as well as in other families (Blattner, 2005; Xiang et al., 2000). However, transoceanic long-distance dispersal across the Pacific Ocean or the Atlantic Ocean cannot be ruled out. Long-distance dispersal across the Atlantic Ocean has been recorded in other genera (Fig. 3, arrow 8; Fritsch, 1996, 2001; Coleman et al., 2001, 2003; Wen and Ickert-Bond, 2009). Analyses of our data suggest that the Hawaiian Islands were colonized from North America (Fig. 2a, node 11) via long-distance dispersal, across the 3900 km oceanic barrier (Fig. 3, arrow 11). On the basis of comparative floristic studies, most natural introductions of Hawaiian flowering plants were probably from Southeastern Asian source areas (Fosberg, 1948). Directionality of prevailing air currents, climatic similarities between the Hawaiian archipelago and Asian tropical areas provide some arguments that support this idea. Since we have not sampled Southeastern Asia completely, we cannot reject a hypothesis of colonization of Hawaii from this area. In contrast, about 20% of ancestral Hawaiian plant colonists are thought to have dispersed from the Americas, despite unfavorable prevailing winds and water currents (Fosberg, 1948; Geiger et al., 2007; Harbaugh et al., 2009). However, long-distance dispersal over the Pacific is confirmed in both cases. A close relationship to the Southern Pacific species Ranunculus caprarum, endemic to the Juan Fernandez archipelago, as hypothesized by Skottsberg (1922), is not supported by our data, because this species is sister to South American taxa (Fig. 2a). 4.2.2. South American–North American disjunctions The South American species Ranunculus maclovianus is sister to the North American Ranunculus orthorhynchus with high bootstrap support in the polyanthemos clade (Fig. 2a, node 10, 100% BS and PP). Based on our biogeographical analyses and previous age estimates, migration from North America to South America has probably occurred in the Pleistocene (Fig. 2a, node 10; Emadzade and Hörandl, in press). Because the position of North and South America has not changed so much since the Cretaceous (Scotese, 2001), flora and fauna could exchange between North and South America through the Isthmus of Panama. On the other hand, disjunctions of plants between the west coast of North America and western South America have been reported several times (Carlquist, 1983; Vargas et al., 1998). The extant widespread distribution of R. orthorhynchus, extending to western North America, makes costal migration of the MRCA by birds more likely (Fig. 3, arrow 10; Wen and IckertBond, 2009). 4.2.3. South American–African disjunction Our data suggest one disjunction event between South America (R. hydrophilus) and Africa (R. meyeri) in the Pleistocene (Fig. 2a, node 2; Emadzade and Hörandl, in press). Although DIVA suggested a vicariance event between South America and Africa, the age of the whole flammula clade (2.9–10.9 My) indicates transoceanic dispersal between these two areas. A vicariance event due to the Gondwana break-up would be 130–100 Myr old (Lomolino et al., 2006). Ranunculus hydrophilus and most other species in this clade occur in wetlands, where birds can be effective for dispersal. Although Lagrange indicated dispersal from Africa to South America, support for this inference was too low to be considered reliable (Fig. 3, arrow 2). Previous studies showed that LDD from South America to South Africa happened in monotypic genera of Ranunculeae (Emadzade and Hörandl, in press), and in other families (Givnish et al., 2004; Schaefer et al., 2009). The African species are restricted to tropical alpine or high mountain habitats (Gehrke and Linder, 2009), and appear in three of the five studied clades (Fig. 2a). There are at least five colonization events of the African high mountains by Ranunculus as suggested by previous studies (Gehrke and Linder, 2009). Due to Author's personal copy 17 K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 addition of more samples from all continents to the data set, our results did not confirm previous results that the African species are nested only within Northern Hemisphere clades (Gehrke and Linder, 2009). Our data show multiple colonizations of the tropical zones from temperate zones of the Southern and the Northern hemisphere. 14 13 15 4.2.4. African–Asian disjunctions A close relationship of African species (Ranunculus volkensii) to Asian species (Ranunculus ficariifolius, distributed in Nepal, Bhutan Sikkim and Thailand) and Ranunculus cheirophyllus (distributed in eastern Asia), is supported well in MP and BI analysis (Fig. 2a, node 4, 59% BS, 100% PP). The current distribution of these taxa and inferences obtained from DIVA suggest that tectonic separation of the Indian subcontinent from Gondwana happened 150 Ma (Raval and Veeraswamy, 2003). However, the very young age of the flammula clade (2.9–10.9 My, Emadzade and Hörandl, in press) makes this hypothesis unlikely. The data are more likely explained by LDD from Asia to Africa, as suggested in ML analyses of Lagrange and parsimony analyses of Mesquite (Fig. 3, arrow 4). (a) Tortonian 14 15 4.2.5. Asian–South American disjunctions Transoceanic dispersal from Eurasia to South America is inferred from the sister relationship of the Mediterranean R. ophioglossifolius and the South American R. flagelliformis in the flammula clade (Fig. 2a, node 3; Fig. 3, arrow 3). Both species occur in wetlands and could have been distributed by birds. Long-distance dispersal over the Atlantic Ocean has been suggested in allied genera and other families with similar distributions as well (e.g., Wendel and Albert, 1992; Coleman et al., 2001, 2003; Tremetsberger et al., 2005; Emadzade and Hörandl, in press). 16 (b) Messinian Eurasia has been colonized by Ranunculus and related genera multiple times during the Neogene (Paun et al., 2005; Hoffmann et al., 2010; Emadzade and Hörandl, in press). Although Eurasian species dominate in two of five clades (Tethyan and arvensis clades), they are present in the other three clades as well. The Eurasian species showed interchange with all other continents through land bridges or transoceanic dispersal (Fig. 3). The Tethyan clade is one of the two clades comprising Eurasian species only (with a few species colonizing also the Mediterranean zone of North Africa, e.g., Ranunculus bullatus). We chose the Tethyan clade to reconstruct the main processes in forming the modern distribution of descendants and effective factors in intracontinental dispersal in greater detail. The origin of the Tethyan clade dates back to the middle Miocene (Fig. 2b, node 13; Emadzade and Hörandl, in press). None of the biogeographical analyses could clearly reconstruct the ancestral area of the Tethyan clade (Fig. 2b, node 13) with high probability; therefore, biogeographical hypotheses remain tentative. If the Mediterranean and the Irano-Turanian region were ancestral areas of this clade, a vicariance event during the Middle Miocene could have isolated the breyninus subclade (Fig. 2b, nodes 13, 15) from its ancestors. This split might have corresponded to the fluctuation of branchings out of the Paratethys and the maximum areal extension of Neo-Paratethys (Fig. 4a; Olteanu and Jipa, 2006). However, a dispersal scenario is also possible. The breyninus subclade is sister to the other members of the Tethyan clade (100% BS, PP), and it is distributed in the high mountain ranges from the Caucasus region to the Alps with its center of morphological diversity in the Caucasus region. The common ancestor of this subclade could have been isolated in the Greater Caucasus until about 5 Ma and then extended its distribution southwards to the Irano-Turanian region after retreat of the Parat- 14 20 4.3. The migration patterns in the Tethyan clade 15 19 18 16 17 (c) Pliocene & Pleistocene Fig. 4. Reconstruction of vicariance and dispersal events in Circum-Mediterranean Ranunculus based on biogeographical studies and dating of events (Emadzade and Hörandl, in press). Arrows and dashed lines depict predominant dispersal and vicariance events, respectively. Numbers in circles referred to nodes of the tree in the Fig. 2b. The distribution of land mass and basins during different periods was based on maps given in Meulenkamp and Sissingh (2003) and Olteanu and Jipa (2006). ethys. In the Pliocene and Pleistocene, Ranunculus breyninus colonized Turkey and the European mountains westwards to the Alps (Fig. 4c). Limitation and migration of ancestors to the Irano-Turanian area took place in the late Miocene (Fig. 2b, nodes 16), which is one of the most interesting stages of the Mediterranean and Paratethyan history. Geographically, this period was characterized by closures of the Betic and Rifian corridors and isolation of the Mediterranean Sea from the Atlantic Ocean, leading to very thick evaporate deposits in the Mediterranean area, known as the ‘‘Messinian Salinity Crisis’’ (Hsü et al., 1973; Hsü et al., 1977; Agustí et al., 2006a, 2006b; Fauquette et al., 2006; Popov et al., 2006; Fig. 4a). The crisis suddenly ended by the ‘‘re-flooding’’ of the Mediterranean basin through the Strait of Gibraltar at the beginning of the Pliocene (Agusti et al., 2006b). After this arid period, the typical summer-dry and winter-wet Mediterranean climate stabilized c. 3 Ma, and an evergreen shrub vegetation established in southern Europe in the late Miocene (Suc, 1984; Willis and McElwain, Author's personal copy 18 K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 2002; Thompson, 2005). In this period, buttercups re-colonized the Eastern Mediterranean area (E. Mediterranean clade, Fig. 2b), and diversified in this area similar to other Mediterranean radiations (e.g., in Anthemis, Lo Presti and Oberprieler, 2009). The diverse ecological conditions of the Mediterranean could have enhanced rapid adaptive radiations (e.g., Guzmán et al., 2009). The separation of the eastern Mediterranean and Irano-Turanian clades could be either explained by vicariance (suggested by DIVA) or by dispersal (suggested by Lagrange and Mesquite). Because of the geographical vicinity and the lack of strong geographical barriers, dispersal is more likely (Fig. 2b, node 19). Our data indicate a closer relationship of the eastern Mediterranean region to the Irano-Turanian ancestors (Fig. 2b, node 19) than to the western Mediterranean buttercup flora. The isolation of areas during the Messinian salinity crisis (Fig. 4b) explains the differentiation between western and eastern part of the Mediterranean species as already observed in Paun et al. (2005). The western Mediterranean clade diversified probably after the onset of the Mediterranean climate in parallel to the eastern radiations, and even reached Macaronesia with one species, R. cortusifolius. All phylogenetic and biogeographical analyses support a close relationship of R. cortusifolius to Western Mediterranean species as observed by Paun et al. (2005), rather than to North African or eastern Mediterranean species (as suggested earlier by Bramwell and Richardson, 1973). Age estimates placed the origin of this species in the late Tertiary, when all of the Canary Islands were already formed (Carracedo, 1994). The initial uplift of the European Alpine system about 10–2 Ma (Plaziat, 1981) provided opportunities for the evolution of alpine taxa. The subalpine-alpine montanus group (Fig. 2b, node 20) originated in the western to central Mediterranean, with at least one migration to the north/meridional to temperate and boreal zones in the Late Pliocene (Fig. 2b, node 20). As the diversification of this montanus clade began at the Plio-Pleistocene period (Fig. 2b, node 20; Emadzade and Hörandl, in press), its radiation could be related to the glaciation cycles of the Quaternary. Similar patterns were recorded in other mountain plant groups (Kadereit and Comes, 2005; Mráz et al., 2007). In contrast to the observations in Anthemis by Lo Presti and Oberprieler (2009), buttercups show a progression from summer-dry to montane humid climates. A multiple parallel colonization of high altitudes occurred in the eastern Irano-Turanian clade. Ranunculus makaluensis originated from an ancestor in the eastern Irano-Turanian region about 3.9 Ma (Fig. 2b, node 17) and dispersed into the high altitudes of Eastern Himalaya, an area that is under the regime of the summer-monsoon. The species is a geographically isolated local endemic of the Makalu glacier region, growing in c. 4000–4500 m altitude (Kadota, 1991), and has no other Himalayan relative in this clade. Therefore, a long-distance dispersal event is likely. The other alpine Himalayan species are nested in clade IV, sister to North American, lowland European, and arctic species (Fig. 1b, clade IV). Therefore, the high altitudes of the Himalayas must have been colonized at least two times independently. In the E. Irano-Turanian clade, Ranunculus elbursensis is confined to the high alpine zones (Iranshahr et al., 1992). Three species colonize the mountain steppes of C. Asia (Ranunculus afghanicus, Ranunculus regelianus, Ranunculus macropodioides), whereby the two former reach the subalpine and alpine zones (Ovczinnikov, 1937). In summary, it is possible to distinguish three periods in the Tethyan clade: (1) colonization of the Mediterranean by a group of species in one (unknown) area in the middle Miocene (Fig. 4a); (2) a putative viariance event during the Messinian, isolating the ancestors of the R. breyninus clade in the Caucasus region and of the western Mediterranean clade (Fig. 4b); (3) range expansions and speciation in the Pliocene and Pleistocene (Fig. 4c), in the west extending to Macaronesia, in the east extending to the Eastern Mediterranean, the Irano-Turanian regions and to Central Asia. Shifts to summer-wet climates in high mountain systems occurred three times independently: in the Alps by R. breyninus and the montanus clade, and in the Himalayas by Ranunculus makaluensis. In all regions (except for the Himalayas), the colonization was followed by a rapid radiation and diversification. 4.4. Capability of long-distance dispersal and rapid adaptation Probably two main features could have made the genus cosmopolitan: successful dispersal over long distances, and establishment and survival in a wide range of habitats. Our data support multiple independent colonizations of different continents. These results reveal that long-distance dispersal may have played an important role for the worldwide distribution of Ranunculus. Three of five clades showed several intercontinental disjunctions within the Northern Hemispheric, Southern Hemispheric and between both hemispheres. The presence of endemic species of Ranunculus in some isolated oceanic islands, for instance on the Hawaii, Juan Fernandez, and Canarian Islands also suggests that LDD in this genus is possible. Long-distance dispersal as an important factor of modern distributions of taxa is recorded in other genera of Ranunculaceae as well (Miikeda et al., 2006; Ehrendorfer et al., 2009; Emadzade and Hörandl, in press). Molecular-based phylogenetic studies based on DNA sequences and estimates of divergence times of lineages supported the role of dispersal as a primary process shaping distribution patterns in both animals and plants (reviewed by de Queiroz, 2005). Achenes in buttercups do not have obvious morphological characters adaptive to dispersal by specific vectors, but Higgins et al. (2003) showed that the relationship between morphological features and long-distance dispersal is poor. Indeed, Green et al. (2008) showed that collected achenes of Ranunculus sceleratus from faecal samples of Anas gracilis successfully germinated. Birds provide well-documented examples of vagrants overcoming distances large enough to explain long-distance dispersal (e.g., Thorup, 1998). Especially the wide distribution of the flammula clade with species adapted to wet habitats can be well explained with dispersal by birds. Local and transoceanic whirlpools could carry simply the small and light achenes of Ranunculus species as well. Indeed, transfer of achenes by wind (anemochory), bird (ornithochory), and water (hydrochory) has been documented in Ranunculus (Müller-Schneider, 1986). Ranunculus could shift to quite different climatic regimes with long-distance and transoceanic dispersal. In the Tethyan clade, shifts from summer-dry climates (Irano-Turanian, Mediterranean) to summer-wet conditions (Alps, Himalayas) happened three times independently (see above). Also in other continents, sister species occur in contrasting climatic regimes, e.g. Ranunculus petiolaris (occurring in the continental climate of southern North America) and Ranunculus hawaiiensis and Ranunculus mauiensis (endemic to subtropical oceanic islands). It seems that Ranunculus has not only a high ability LDD to new areas but also to rapid adaptation to new habitats. In summer-dry climates, buttercups have developed special morphological adaptations like tuberous roots (Tamura, 1995; Paun et al., 2005). In the subtropical and tropical zones, they shift to wetlands (e.g., R. petiolaris) or to high altitudes (e.g., the African or Himalayan species). In contrast, species colonizing the Arctic do not have any obvious novel morphological features, probably because of a pre-adaptation of buttercups to wet habitats (Hoffmann et al., 2010). Establishment after long dispersal may be enhanced by different reproductive strategies, such as vegetative propagation, self-compatibility and agamospermy (Hörandl et al., 2005; Hörandl, 2008). Moreover, buttercups have generalist flowers and therefore a broad spectrum of pollinators (Steinbach and Gottsberger, 1994), which may help for establishment in new environments. Author's personal copy K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 However, the high potential for dispersal and colonization of new areas are not the only factors for the biogeographical history of Ranunculus. In the Tethyan clade, we exemplify that geographical isolation is often followed by rapid speciation, probably due to strong adaptive radiations. Ecological differentiation into various micro-niches was recognized as a major factor for speciation in the Ranunculus montanus clade (Dickenmann, 1982). Polyploidy and hybridization can further contribute to sympatric speciation and diversification (Hörandl et al., 2005). Previous studies based on ITS sequence data on a larger regional sampling indicated major radiations in New Zealand (Lockhart et al., 2001; Lehnebach, 2008), Southern South America, Australia, and in the Malesian mountains (Hörandl et al., 2005; Lehnebach, 2008) similar to those in the Tethyan clade. The species-richness and the cosmopolitan distribution of Ranunculus are probably caused by the interplay of transoceanic plus intracontinental dispersal, and a potential for rapid adaptation and speciation. Acknowledgments We are grateful to D. Albach, M. Ghahremanii, J.T. Johansson, C. Keener, C. Lehnebach, F. Lone, M. Mirtajeddini, H. Maroofi, C. Rebernig, K. Safikhani, G. Schneeweiss, P. Schönswetter, T.F. Stuessy, and A. Tribsch for collecting materials, N. Tkach for some data from Ranunculus s.str., the curators of the herbaria BISH, CAN, CONN, GB, LD, LE, LI, M, MPN, RM, TARI, VALD, WU, W, ZH and ZT for the loan of herbarium specimens and permission to use materials for DNA extractions. The authors are grateful to the Commission for Interdisciplinary Ecological Studies (KIÖS) of the Austrian Academy of Sciences (ÖAW), the Austrian Science Fund (FWF), Project I 310, and the National Geographic Society (Project 8773-08) for Grants to E.H., and the Austrian Exchange Service (ÖAD) for a Ph.D. student Grant to K.E. The suggestions of two anonymous reviewers have been of great value. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev.2010.11.002. References Agustí, J., Garcés, M., Krijgsman, W., 2006a. Evidence for African–Iberian exchanges during the Messinian in the Spanish mammalian record. Palaeogeogr., Palaeoclimatol., Palaeoecol. 238, 5–14. Agusti, J., Oms, O., Meulenkamp, J.E., 2006b. Introduction to the Late Miocene to Early Pliocene environment and climate change in the Mediterranean area. Palaeogeogr., Palaeoclimatol., Palaeoecol. 238, 1–4. Blattner, F.R., 2005. Multiple intercontinental dispersals shaped the distribution area of Hordeum (Poaceae). New Phytol. 169, 603–614. Bramwell, D., Richardson, I.B.K., 1973. Floristic connections between Macaronesia and the East Mediterranean region. Monogr. Biol. Canarienses 4, 118–125. Carlquist, S., 1983. Intercontinental dispersal. Sonderbd. Naturwiss. Ver. Hamburg 7, 37–47. Carracedo, J.C., 1994. The Canary Islands: an example of structural control on the growth of large oceanic-island volcanoes. J. Volcanol. Geotherm. Res. 60, 225– 241. Coleman, M., Forbes, D.G., Abbott, R.J., 2001. A new subspecies of Senecio mohavensis (Compositae) reveals Old–New World species disjunction. Edinb. J. Bot. 58, 389–403. Coleman, M., Liston, A., Kadereit, J.W., Abbott, R.J., 2003. Repeat intercontinental dispersal and Pleistocene speciation in disjunct Mediterranean and desert Senecio (Asteraceae). Am. J. Bot. 90, 1446–1454. Cook, J.A., Hoberg, E.P., Koehler, A., Henttonen, A., Wickström, L., Haukisalmi, V., Galbreath, K., Chernyavski, F., Dokuchaev, N., Lahzuhtkin, A., MacDonald, S.M., Hope, A., Waltari, E., Runck, A., Veitch, A., Popko, R., Jenkins, E., Kutz, S., Eckerlin, R., 2005. Beringia: intercontinental exchange and diversification of high latitude mammals and their parasites during the Pliocene and Quaternary. Mamm. Study 30, S33–S44. Davis, C.C., Fritsch, P.W., Li, J., Donoghue, M.J., 2002. Phylogeny and biogeography of Cercis (Fabaceae): evidence from nuclear ribosomal ITS and chloroplast ndhF sequence data. Syst. Bot. 27, 289–302. 19 de Queiroz, A., 2005. The resurrection of oceanic dispersal in historical biogeography. Trends Ecol. Evol. 20, 68–73. Dickenmann, R., 1982. Genetic-ecological investigations in Ranunculus montanus Willd. s.l. from the alpine vegetation belt of Davos (Grisons). Veröff. Geobot. Inst. ETH Stiftung Rübel, Zürich, 78, 1–89 (German with English abstract). Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. Bot. Soc. Am. 19, 11–15. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Ehrendorfer, F., Ziman, S.N., König, C., Keener, C.S., Dutton, B.E., Tsarenko, O.N., Bulakh, E.V., Bosßcaiu, M., Médail, F., Kästner, A., 2009. Taxonomic revision, phylogeny and transcontinental distribution of Anemone section Anemone (Ranunculaceae). Bot. J. Linn. Soc. 160, 312–354. Emadzade, K., Hörandl, E, in press. Northern Hemisphere origin, transoceanic dispersal, and diversification of Ranunculeae, DC. (Ranunculaceae) in the Cenozoic. J. Biogeogr. doi:10.1111/j.1365-2699.2010.02404.x. Emadzade, K., Lehnebach, C., Lockhart, P., Hörandl, E., 2010. A molecular phylogeny, morphology and classification of genera of Ranunculeae (Ranunculaceae). Taxon 59, 809–828. Fauquette, S., Suc, J.-P., Bertini, A., Popescu, S.-M., Warny, S., Bachiri Taoufiq, N., Perez Villa, M.-J., Chikhi, H., Feddi, N., Subally, D., Clauzon, G., Ferrier, J., 2006. How much did climate force the Messinian salinity crisis? Quantified climatic conditions from pollen records in the Mediterranean region. Palaeogeogr., Palaeoclimatol., Palaeoecol. 238, 281–301. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Fitch, W.M., 1971. Toward defining the course of evolution: minimal change for a specific tree topology. Syst. Zool. 20, 406–416. Fosberg, F.R., 1948. Introduction. In: Zimmerman, E.C. (Ed.), Insects of Hawaii. Hawaii Univ. Press, Honolulu, HI, pp. 107–119. Fritsch, P., 1996. Isozyme analysis of intercontinental disjuncts within Styrax (Styracaceae): implications for the Madrean–Tethyan hypothesis. Am. J. Bot. 83, 342–355. Fritsch, P., 2001. Phylogenetics and biogeography of the flowering plant genus Styrax (Styracaceae) based on chloroplast DNA restriction sites and DNA sequences of the internal transcribed spacer region. Mol. Phylogenet. Evol. 19, 387–408. Gehrke, B., Linder, H.P., 2009. The scramble for Africa: pan-temperate elements on the African high mountains. Proc. R. Soc. B 276, 2657–2665. Geiger, J.M.O., Ranker, T.A., Neale, J.M.R., Klimas, S.T., 2007. Molecular biogeography and origins of the Hawaiian fern flora. Brittonia 59, 142–158. Givnish, T.J., Millam, K.C., Evans, T.M., Hall, J.C., Pires, J.C., Berry, P.E., Sytsma, K.J., 2004. Ancient vicariance or recent long-distance dispersal? Inferences about phylogeny and South American–African disjunctions in Rapateaceae and Bromeliaceae based on ndhF sequence data. Int. J. Plant Sci. Suppl. 165, 35–54. Green, A.J., Jenkins, K.M., Bell, D., Morris, P.J., Kingsford, R.T., 2008. The potential of waterbirds in dispersing invertebrates and plants in arid Australia. Freshw. Biol. 53, 380–392. Gruenstaeudl, M., Urtubey, E., Jansen, R.K., Samuel, R., Barfuss, M.H., Stuessy, T.F., 2009. Phylogeny of Barnadesioideae (Asteraceae) inferred from DNA sequence data and morphology. Mol. Phylogenet. Evol. 51, 572–587. Guzmán, B., Dolores Lledó, N., Vargas, P., 2009. Adaptive radiation in mediterranean Cistus (Cistaceae). PLoS ONE 4, e6362. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95– 98. Harbaugh, D.T., Wagner, W.L., Allan, G.J., Zimmer, E.A., 2009. The Hawaiian Archipelago is a stepping stone for dispersal in the Pacific: an example from the plant genus Melicope (Rutaceae). J. Biogeogr. 36, 230–241. Higgins, S.I., Nathan, R., Cain, M.L., 2003. Are long-distance dispersal events in plants usually caused by nonstandard means of dispersal? Ecology 84, 1945– 1956. Hileman, L.C., Vasey, M.C., Parker, V.T., 2001. Phylogeny and biogeography of the Arbutoideae (Ericaceae): implications for the Madrean–Tethyan hypothesis. Syst. Bot. 26, 131–143. Hoffmann, M.H., von Hagen, K.B., Hörandl, E., Röser, M., Tkach, N.V., 2010. Sources of the arctic flora: origins of arctic species in Ranunculus and related genera. Int. J. Plant Sci. 171, 90–106. Hoot, S.B., 1994. Phylogeny of the Ranunculaceae based on preliminary atpB, rbcL, and 18S nuclear ribosomal DNA sequence data. Plant Syst. Evol. Suppl. 9, 241– 251. Hopkins, D.M., 1959. Cenozoic history of the Bering Land Bridge. Science 129, 1519– 1528. Hörandl, E., 2008. Evolutionary implications of self-compatibility and reproductive fitness in the apomictic Ranunculus auricomus polyploid complex (Ranunculaceae). Int. J. Plant Sci. 169, 1219–1228. Hörandl, E., Emadzade, K., in press. The evolution of alpine species in Ranunculus (Ranunculaceae) – A global comparison. Taxon 60. Hörandl, E., Paun, O., Johansson, J.T., Lehnebach, C., Armstrong, T., Chen, L., Lockhart, P., 2005. Phylogenetic relationships and evolutionary traits in Ranunculus s.l. (Ranunculaceae) inferred from ITS sequence analysis. Mol. Phylogenet. Evol. 36, 305–327. Hörandl, E., Greilhuber, J., Klimova, K., Paun, O., Temsch, E., Emadzade, K., Hodálová, I., 2009. Reticulate evolution and taxonomic concepts in the Ranunculus auricomus complex (Ranunculaceae): insights from morphological, karyological and molecular data. Taxon 58, 1194–1215. Author's personal copy 20 K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 Hsü, K.J., Ryan, W.B.F., Cita, M.B., 1973. Late Miocene desiccation of the Mediterranean. Nature 242, 240–244. Hsü, K.J., Montadert, L., Bernoulli, D., Cita, M.B., Erickson, A., Garrison, R.E., Kidd, R.B., Mèlierés, F., Müller, C., Wright, R., 1977. History of the Messinian salinity crisis. Nature 267, 399–403. Huber, B.A., 2003. On the terminology of polytomies. Cladistics 19, 273. Hulten, E., 1937. Outline of the History of Arctic and Boreal Biota During Quaternary Period. Bokforlags Aktiebolgaet, Thule, Stockholm. Iranshahr, M., Rechinger, K.H., Riedl, H., 1992. Ranunculaceae. Flora Iranica. Rechinger, K.H. (Ed.), Akad. Druck- u. Verlagsanstalt, Graz., vol. 171, pp. 1–249. Johansson, J.T., 1995. A revised chloroplast DNA phylogeny of the Ranunculaceae. Plant Syst. Evol. Suppl. 9, 253–261. Johansson, J.T., 1998. Chloroplast DNA restriction site mapping and the phylogeny of Ranunculus (Ranunculaceae). Plant Syst. Evol. 213, 1–19. Kadereit, J.W., Comes, H.P., 2005. The temporal course of alpine plant diversification in the Quaternary. In: Bakker, F.T., Chatrou, L.W., Gravendeel, B., Pelser, P. (Eds.), Plant Species-level Systematics: New Perspectives on Pattern and Process. A.R.G. Gantner Verlag, Ruggell, Liechtenstein, pp. 117–130. Kadota, Y., 1991. Taxonomic Notes on Some Alpine Species of Ranunculus (Ranunculaceae) in the Himalaya. Univ. Mus. Tokyo Bull., p. 34. Kodandaramaiah, U., 2010. Use of dispersal–vicariance analysis in biogeography – a critique. J. Biogeogr. 37, 3–11. Kron, K.A., Luteyn, J.L., 2005. Origins and biogeographic patterns in Ericaceae: new insights from recent phylogenetic analyses. In: Friis, I., Balslev, H. (Eds.), Plant Diversity and Complexity Patterns. Det Kongelige Dansk Videnskaberneses Selskap, Copenhagen, Denmark, pp. 479–500. Lehnebach, C.A., 2008. Phylogenetic affinities, species delimitation and adaptive radiation of New Zealand Ranunculus. Ph.D. thesis, Massey University, Palmerston North, New Zealand. Lehnebach, C.A., Cano, A., Monsalve, C., McLenachan, P., Hörandl, E., Lockhart, P., 2007. Phylogenetic relationships of the monotypic Peruvian genus Laccopetalum (Ranunculaceae). Plant Syst. Evol. 264, 109–116. Liston, A., 1997. Biogeographic relationships between the Mediterranean and North American floras: insights from molecular data. Lagascalia 19, 323–330. Lo Presti, R.M., Oberprieler, C., 2009. Evolutionary history, biogeography and ecoclimatological differentiation of the genus Anthemis L. (Compositae, Anthemideae) in the circum-Mediterranean area. J. Biogeogr. 136, 1313–1332. Lockhart, P., McLechnanan, P.A., Havell, D., Glenny, D., Huson, D., Jensen, U., 2001. Phylogeny, dispersal and radiation of New Zealand alpine buttercups: molecular evidence under split decomposition. Ann. Miss. Bot. Garden 88, 458–477. Lomolino, M.V., Riddle, B.R., Brown, J.H., 2006. Biogeography, third ed. Sinauer Associates Inc., Sunderland. Lourteig, A., 1984. Ranunculaceae. In: Correa, M.N. (Ed.), Flora Patagonica, vol. IV-a. Buenos Aires, pp. 284–321. Maddison, W.P., Maddison, D.R., 2009. MESQUITE: A Modular System for Evolutionary Analysis. 2.6. <http://mesquiteproject.org>. Manos, P.S., Donoghue, M.J., 2001. Progress in northern hemisphere phytogeography: an introduction. Int. J. Plant Sci. Suppl. 162 (6), 1–2. Meulenkamp, J.E., Sissingh, W., 2003. Tertiary palaeogeography and tectonostratigraphic evolution of the Northern and Southern Peri-Tethys platforms and the intermediate domains of the African–Eurasian convergent plate boundary zone. Palaeogeogr., Palaeoclimatol., Palaeoecol. 196, 209–228. Meusel, W., Jäger, E., Weinert, E., 1965. Chorologie der Zentraleuropäischen Flora. G. Fischer, Jena. Miikeda, O., Kita, K., Handa, T., Yukawa, T., 2006. Phylogenetic relationships of Clematis (Ranunculaceae) based on chloroplast and nuclear DNA sequences. Bot. J. Linn. Soc. 152, 153–168. Mráz, P., Gaudeul, M., Rioux, D., Gielly, L., Choler, P., Taberlet, P., 2007. Genetic structure of Hypochaeris uniflora (Asteraceae) suggests vicariance in the Carpathians and rapid post-glacial colonization of the Alps from an eastern Alpine refugium. J. Biogeogr. 34, 2100–2114. Müller, K., 2005. SeqState – primer design and sequence statistics for phylogenetic DNA data sets. Appl. Bioinformatics 4, 65–69. Müller-Schneider, P., 1986. Verbreitungsbiologie der Blütenpflanzen Graubündens, diasporology of the spermatophytes of the grisons (Switzerland). Veröffentlichungen des Geobotanischen. Insitutes der ETH, Stiftung Rübel, Zürich 85, 1–263. Nylander, J.A.A., 2004. MrModeltest v2. Program Distributed by the Author. Evolutionary Biology Centre, Uppsala University. Olteanu, R., Jipa, D.C., 2006. Dacian basin environmental evolution during upper neogene within the Paratethys domain. GeoEcoMarina 12, 91–106. Ovczinnikov, P.N., 1937. Ranuculus. In: Komarov, W.A. (Ed.), Flora URSS, vol. VII. Ranales and Rhoeadales. Botanicheskii Institut Akademii Nauk USSR, Moscow, pp. 351–509 (English Translation 1970). Paun, O., Lehnebach, C., Johansson, J.T., Lockhart, P., Hörandl, E., 2005. Phylogenetic relationships and biogeography of Ranunculus and allied genera (Ranunculaceae) in the Mediterranean region and in the European alpine system. Taxon 54, 911–930. Plaziat, J.C., 1981. Late Cretaceous to Late Eocene paleogeographic evolution of southwest Europe. Palaeogeogr., Palaeoclimatol., Palaeoecol. 36, 263–320. Popov, S.V., Shcherba, I.G., Ilyina, L.B., Nevesskaya, L.A., Paramonova, N.P., Khondkarian, S.O., Magyar, I., 2006. Late Miocene to Pliocene palaeogeography of the Paratethys and its relation to the Mediterranean. Palaeogeogr., Palaeoclimatol., Palaeoecol. 238, 91–106. Rambaut, A., Drummond, A., 2002. Tree Annotator Version 1.4 Part of the BEAST Package. <http://evolve.zoo.ox.ac.uk/>. Rau, M.A., 1993. Ranunculaceae. In: Sharma, B.D., Balakrishnan, N.P., Rao, R.R., Hajra, P.K. (Eds.), Flora of India, vol. 1. Botanical survey of India, Calcutta, pp. 1–145. Raval, U., Veeraswamy, K., 2003. India–Madagascar separation: breakup along a pre-existing mobile belt and chipping of the Craton. Gondwana Res. 6, 467–485. Ree, R.H., Smith, S.A., 2008. Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis. Syst. Biol. 57, 4–14. Ree, R.H., Moore, M.R., Webb, C.O., Donoghue, M.J., 2005. A likelihood framework for inferring the evolution of geographic range on phylogenetic trees. Evolution 59, 2299–2311. Riedl, H., Nasir, Y., 1990. Ranunculaceae. In: Ali, S.I., Nasir, Y.J. (Eds.), Flora of Pakistan, vol. 193. PanGraphics, Islamabad. Ro, K., Keener, C.S., McPheron, B.A., 1997. Molecular phylogenetic studies of the Ranunculaceae: utility of the nuclear 26S ribosomal DNA in inferring intrafamiliar relationships. Mol. Phylogenet. Evol. 8, 117–127. Ronquist, F., 1996. DIVA Version 1.1. Computer Program and Manual Available by Anonymous FTP from Uppsala University. Ronquist, F., 1997. Dispersal–vicariance analysis: a new approach to the quantification of historical biogeography. Syst. Biol. 46, 195–203. Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Schaefer, H., Heibl, C., Renner, S.S., 2009. Gourds afloat: a dated phylogeny reveals an Asian origin of the ground family (Cucurbitaceae) and numerous oversea dispersal events. Proc. R. Soc. B 276, 843–851. Scotese, C.R., 2001. Atlas of Earth History, vol. 1. PALEOMAP Project. Arlington, Texas, USA. Shaw, J., Lickey, E.B., Schilling, E.E., Small, R.L., 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. Am. J. Bot. 94, 275–288. Simmons, M.P., Ochoterena, H., 2000. Gaps as characters in sequence-based phylogenetic analyses. Syst. Biol. 49, 369–381. Skottsberg, C., 1922. Ranunculus caprarum. In: Skottsberg, C. (Ed.), The Natural History of Juan Fernandez and Easter Island, Part 2. Almqvist & Wiksells, Uppsala, Sweden, pp. 123–124. Steinbach, K., Gottsberger, G., 1994. Phenology and pollination biology of five Ranunculus species in Giessen, Central Germany. Phyton 34, 203–218. Suc, J.P., 1984. Origin and evolution of the Mediterranean vegetation and climate in Europe. Nature 307, 429–432. Sullivan, J., Swofford, D.L., 2001. Should we use model-based methods for phylogenetic inference when we know that assumptions about among-site rate variation and nucleotide substitution pattern are violated? Syst. Biol. 50, 723–729. Swofford, D.L., 2002. PAUP; Phylogenetic Analysis Using Parsimony (and Other Methods). Ver. 4.0b. 8. Sinauer Associates, Sunderland, MA, USA. Takhtajan, A., 1986. Floristic Regions of the World. University of California Press, Berkeley, USA. Tamura, M., 1993. Ranunculaceae. In: Kubitzki, K., Rohwer, J.G., Bittrich, V. (Eds.), The Families and Genera of Vascular Plants. 2. Flowering Plants. Dicotyledons, Magnoliid, Hamamelid, and Caryophyllid Families. Springer, Berlin, pp. 563–583. Tamura, M., 1995. Angiospermae. Ordnung Ranunculales. Fam. Ranunculaceae. II. Systematic Part. In: Hiepko, P. (Ed.), Natürliche Pflanzenfamilien, second ed., vol. 17aIV. Duncker & Humblot, Berlin, pp. 223–519. Tel-Zur, N., Abbo, S., Myslabodski, D., Mizrahi, Y., 1999. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Mol. Biol. Rep. 17, 249–254. Thompson, J.D., 2005. Plant Evolution in the Mediterranean. Oxford University Press, Oxford. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgings, D.G., 1997. The clustral X Windows interface. Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882. Thorup, K., 1998. Vagrancy of yellow-browed Phylloscopus inornatus and Pallas’ warbler Ph. proregulus in north-west Europe: misorientation on great circles? Ringing Migr. 19, 7–12. Tiffney, B.H., 2000. Geographic and classification influences on the Cretaceous and Tertiary history of Euramerican floristic similarity. Acta Univ. Carol. Geol. 44, 5– 16. Tiffney, B.H., Manchester, R.S., 2001. The use of geological and paleontological evidence in evaluating plant phylogeographic hypotheses in the northern hemisphere Tertiary. Int. J. Plant. Sci. 162, S3–S17. Tremetsberger, K., Weiss-Schneeweiss, H., Stuessy, T.F., Samuel, R., Kadlec, G., Ortiz, M.A., Talavera, S., 2005. Nuclear ribosomal DNA and karyotypes indicate a NW African origin of South American Hypochaeris (Asteraceae, Cichorieae). Mol. Phylogenet. Evol. 35, 102–116. Tutin, T.G., Cook, C.D.K., 1993. Ranunculus. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea I: Psilotaceae to Platanaceae, second ed. Cambridge Univ. Press, Cambridge, pp. 269–290. Vargas, P., Baldwin, B., Constance, L., 1998. Nuclear ribosomal DNA evidence for a western North American origin of Hawaiian and South American species of Sanicula (Apiaceae). PNAS 95, 235–240. Wen, J., 1999. Evolution of eastern Asian and eastern North American disjunct distributions in flowering plants. Ann. Rev. Ecol. Syst. 30, 421–455. Wen, J., Ickert-Bond, S.M., 2009. Evolution of the Madrean–Tethyan disjunctions and the North and South American amphitropical disjunctions in plants. J. Syst. Evol. 47, 331–348. Wencai, W., Gilbert, M.G., 2001. Ranunculus. In: Flora China, vol. 6. Science Press, Beijing; Missouri Botanical Garden Press, St. Louis, pp. 391–431. Author's personal copy K. Emadzade et al. / Molecular Phylogenetics and Evolution 58 (2011) 4–21 Wendel, J., Albert, V., 1992. Phylogenetics of the cotton genus (Gossypium): character state weighted parsimony analysis of chloroplast-DNA restriction site data and its systematic and biogeography implications. Syst. Bot. 17, 115– 143. Whittemore, A., 1997. Ranunculus. In: Flora of North America Committee (Eds.), Flora of North America, North of Mexico, vol. 3. Magnoliophyta: Magnoliidae and Hamamelidae. Oxford Univ. Press, New York, pp. 88–135. 21 Willis, K.J., McElwain, J.C., 2002. The Evolution of Plants. Oxford Univ. Press, New York. Xiang, Q.-Y., Soltis, D.E., Soltis, P.S., Manchester, S.R., Crawford, D.J., 2000. Timing the Eastern Asian–Eastern North American floristic disjunction: molecular clock corroborates paleontological estimates. Mol. Phylogenet. Evol. 15, 462–472. Yurtsev, B.A., 1974. Problems of the Botanical Geography of Northeast Asia. Nauka, Leningrad, Russia.

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