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Evidence for a vicariant origin of Macaronesian–Eritreo/Arabian disjunctionsin Campylanthus Roth (Plantaginaceae)

Mike Thiv a,*, Mats Thulin b, Mats Hjertson c, Matthias Kropf d, Hans Peter Linder e

a Botany Department, Staatliches Museum für Naturkunde, Rosenstein 1, D-70191 Stuttgart, Germanyb Department of Evolution, Genomics and Systematics, University of Uppsala, Norbyvägen 18D, 75236 Uppsala, Swedenc Museum of Evolution, University of Uppsala, Norbyvägen 16, 75236 Uppsala, Swedend Institut für Botanik, Universität für Bodenkultur, Gregor Mendel-Str. 33, A-1180 Vienna, Austriae Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, CH 8008 Zurich, Switzerland

a r t i c l e i n f o

Article history:Received 26 May 2009Revised 5 October 2009Accepted 6 October 2009Available online 13 October 2009

Keywords:CampylanthusPlantaginaceaeBiogeographyPhylogenyVicarianceTrans-Saharan disjunctionMacaronesia

a b s t r a c t

The numerous disjunct plant distributions between Macaronesia and eastern Africa–Arabia suggest thatthese could be the relicts of a once continuous vegetation belt along the southern Tethys, which has beenfragmented by Upper Miocene–Pliocene aridification. We tested this vicariance hypothesis with a phylo-genetic analysis of Campylanthus (Plantaginaceae), based on nuclear and plastid DNA sequence data. Ourresults indicate a basal split within Campylanthus giving rise to Macaronesian and Eritreo–Arabian lin-eages in the Pliocene/Upper Miocene. This is consistent with the vicariance hypothesis, thus obviatingthe need to postulate trans-Saharan long-distance dispersal. The biogeography of Campylanthus may par-allel patterns in other plant groups and the implications for our understanding of the biogeography ofnorthern and eastern Africa, and Arabia are discussed.

Crown Copyright � 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction

Disjunct distribution patterns are common in many organismgroups, and their patterns and causes have early gained theinterest of natural scientists (e.g., Darwin, 1859). Two main mech-anisms resulting in a fragmented distribution can be distinguished.Vicariance results from an emerging barrier splitting a continuousrange into two or more separate parts, while in case of (long-dis-tance) dispersal disjunct distribution areas are the result of dis-persal over pre-existing barriers (Nelson, 1978; de Queiroz,2005). The relative importance of these processes has been inten-sively debated in the last decades. Until a decade ago vicarianceexplanations were preferred, as they result in more generalhypotheses or explanations for similar disjunctions across differentorganismic groups. Moreover, vicariance hypotheses were falsifi-able, for example if the phylogeny of a taxon was incongruent withthe hypothesised vicariance history. Vicariance resulting fromearth history events, such as plate tectonics, could affect wholebiotas, while dispersal was considered to be mainly taxon-specific.

Consequently, dispersal was only accepted a priori for cases with-out a vicariance explanation, e.g., for the colonisation of volcanicoceanic islands. In recent decades long-distance dispersal hasemerged again as the most popular process for shaping current dis-junct distributions (e.g., Renner, 2004; de Queiroz, 2005; Keppelet al., 2009). This has largely been the result of molecular datingtechniques, which often falsified the vicariance prediction thatthe divergence of the variant taxa should be at least as old as thegeological event that caused the disjunction (e.g., plate move-ments, mountain range formations; de Queiroz, 2005).

A typical example of a disjunction is between eastern Africa–Arabia and the Macaronesian Islands (in a traditional sense com-prising the Canary Islands, Madeira, Cape Verde Islands, Azoresand Salvage Islands, of which, however, the Cape Verde Islandswere shown to have closer links to tropical Africa, Vanderpoortenet al., 2007, see discussion; Bramwell, 1976; Quézel, 1978; Deiland Müller-Hohenstein, 1984; Mies, 1995). Examples includeCampylanthus Roth (Plantaginaceae), Dracaena L. (Dracaenaceae/Ruscaceae), Hemicrambe Webb (Brassicaceae), Parolinea Webb(Brassicaceae), Aeonium Webb & Berthel. (Crassulaceae), Camptolo-ma Benth. (Scrophulariaceae), and Pulicaria L./Vieraea Sch. Bip.(Asteraceae; Andrus et al., 2004). These disjunctions have beeninterpreted as relicts of a late Miocene continuous, subtropicalflora in northern Africa (Hooker, 1878; Engler, 1879; Meusel,

1055-7903/$ - see front matter Crown Copyright � 2009 Published by Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2009.10.009

* Corresponding author. Fax: +49 (0) 711 8936100.E-mail addresses: thiv.smns@naturkundemuseum-bw.de (M. Thiv), Mats.Thuli-

n@ebc.uu.se (M. Thulin), Mats.Hjertson@evolmuseum.uu.se (M. Hjertson), matthia-s.kropf@boku.ac.at (M. Kropf), peter.linder@systbot.uzh.ch (H.P. Linder).

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1965; Axelrod, 1975; Sunding, 1979; Bramwell, 1985; Thiede,1994; Thulin, 1994; Marrero et al., 1998). This vegetation beltwas fragmented by aridification in the Upper Miocene/Pliocene/Quaternary that resulted in the formation of the Sahara and theconsequent extinction of many plant groups in this area. Some spe-cies of a few members of this flora survived in the Canary Islandsand Arabia/eastern Africa, leading to the present day disjunct pat-terns (Sunding, 1979; Bramwell, 1985). This is a typical vicariancescenario and is therefore referred to as the vicariance hypothesis.Such biogeographic implications have mostly been inferred fromtaxonomy-based distribution patterns. Only a few molecular phy-logenetic studies addressing this question have been conducted,and these confirmed a sister group relationship between the Mac-aronesian and eastern African/Arabian species for Camptoloma,Aeonium and Plocama (Kornhall et al., 2001; Mort et al., 2002;Backlund et al., 2007). Most taxa, however, do not fit the vicariancehypothesis (e.g., Pulicaria/Vieraea, Andrus et al., 2004), as the clos-est relatives of the Macaronesian species are not found in East Afri-ca, but in the Mediterranean basin.

Here, we test the vicariance hypothesis using a prominentexample of Macaronesian–Eritreo/Arabian disjunctions, thepalaeotropical genus Campylanthus (Plantaginaceae, formerlyScrophulariaceae). Campylanthus includes 18 species of shrubsand subshrubs, two being restricted to Macaronesia (Canary andCape Verde Islands) and 16 occurring from east Africa, Arabia, toPakistan (Fig. 1, Miller, 1980, 1982, 1988; Hjertson, 1997, 2003;Hjertson and Miller, 2000; Kilian et al., 2002; Hjertson and Thulin,2006; Hjertson et al., 2008). We used a molecular phylogeneticanalysis and a molecular dating approach employing a relaxed

molecular clock, to address the following questions: (1) Do theMacaronesian and Eritreo–Arabian taxa constitute sister clades assuggested by cladistic analyses based on morphological data(Hjertson, 1997, 2003), thus consistent with a vicariance scenario?(2) If so, does the age of the divergence between the Eritreo–Ara-bian and Macaronesian clades coincide with the late Miocene onsetof Saharan aridification, as predicted by the vicariance hypothesis?(3) Finally, we aim at reconstructing the biogeographic history ofCampylanthus. To this end, we analysed sequence data of the plas-tid (atpB-rbcL intergenic spacer) and the nuclear genomes (nrITS,Baldwin et al., 1995; Alvarez and Wendel, 2003) from nearly allcurrently recognised Campylanthus taxa and used a Bayesian re-laxed clock approach to date relevant nodes.

2. Material and methods

2.1. Taxon sampling

The genus Campylanthus consists of 18 species, 14 of whichwere included in our analyses (Table 1). The remaining four – theSomalian C. anisotrichus and C. parviflorus, the Omanian C. hajaren-sis and the Pakistani C. ramosissimus – are only rarely collected dueto their very restricted distribution and, therefore, no material wasavailable for our study. The position of Campylanthus within theformer Scrophulariaceae was unresolved (Reichenbach, 1828; Ben-tham and Hooker, 1886; Wettstein, 1891; Hjertson, 1997, 2003;Fischer, 2004) until recent molecular phylogenetic analyses (Al-bach et al., 2005) placed it as sister to the Globularia L./Poskea Vatke

Table 1Taxon sampling, vouchers, defined areas of endemism and Genbank numbers: MA, Macaronesian Region; SA, South Arabian Province; SE, Somalo-Ethiopian Province; SO,Socotran Province. Sequences marked with an asterisk were used for Bayesian dating.

Taxon Voucher Herbarium Origin Defined area ofdistribution

nrITS atpB-rbcLspacer

Aragoa abietina H.B. & K. See reference AJ459404*

Aragoa cupressina H.B. & K. See reference AJ459402*

Campylanthus antonii Thulin Thulin et al. 9534 UPS Yemen SA FM207412* FM207432Campylanthus chascaniflorus A.G. Mill. Miller 7731 E Oman SA FM207413* FM207433Campylanthus glaber Benth. Kilian 3140 B Cape Verde Islands MA FM207414* FM207434Campylanthus hubaishanii N. Kilian &

P. HeinKilian YP1169 B Yemen SA FM207416* FM207435

Campylanthus incanus A.G. Mill. Thulin et al. 10720 UPS Somalia SE FM207415* FM207436Campylanthus junceus Edgew. Kilian 6036 B Yemen SA, SE FM207417* FM207437Campylanthus mirandae A.G. Mill. Morris 561 E Oman SA FM207418* FM207438Campylanthus pungens O. Schwartz Kilian 5138 B Yemen SA FM207419* FM207439Campylanthus salsoloides (L.f.) Roth Wieringa & Janzen

3485WAG Spain (Canary

Islands)MA FM207421* FM207441

Campylanthus sedoides A.G. Mill. Miller & Nyberg 9451 BM Oman SA FM207422* FM207447Campylanthus somaliensis A.G. Mill. Thulin & Warfa 5560 UPS Somalia SE FM207425* FM207442Campylanthus reconditus Hjertson &

ThulinThulin et al. 10652 UPS Somalia SE FM207420* FM207440

Campylanthus spinosus Balf. (f.) Kilian 2374 B Yemen (Socotra) SO FM207426* FM207443Campylanthus spinosus Balf. (f.) Lavranos & Carter

23477WAG Somalia SE FM207427* FM207444

Campylanthus yemenensis A.G. Mill. Miller 3076 E Yemen SA FM207423* FM207445Campylanthus yemenensis A.G. Mill. Wood 75/225 E Yemen SA FM207424* FM207446Digitalis lutea L. Brune s.n. STU Germany FM207428Digitalis lutea L. See reference AY591266*

Globularia punctata Lapeyr. Joßberger & Brune s.n. STU Germany FM207431Globularia repens Lam. See reference AY492105*

Globularia salicina Lam. See reference AF313039*

Plantago major L. See reference AY101861*

Plantago major L. Joßberger & Brune s.n. STU Germany FM207430Plantago raoulii Decne. See reference AY101867*

Plantago stauntonii Reichardt See reference AY101870*

Plantago debilis R. Br. See reference AY101868*

Plantago spathulata Hook. f. See reference AY101869*

Plantago atrata Hoppe See reference AY101895*

Plantago coronopus L. See reference AY101882*

Plantago uniflora L. See reference AY101885*

Veronica filiformis Sm. Joßberger s.n. STU Germany GU143559 FM207429

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clade. The clade Campylanthus, Globularia, Poskea, in turn, belongsto a larger lineage including, among others, Plantago L., VeronicaL., and Digitalis L. Accordingly, we chose members of Digitalis,Veronica, Plantago and Globularia as outgroups for the combineddata set. Due to lack of material, we merged plastid and nuclearDNA sequences of G. punctata and G. repens to represent a singlechimeric member of Globularia. For molecular dating, a modifiednrITS data set was used by adding more species of Plantago andAragoa Kunth (Bello et al., 2002) to the taxon sample, which wererelevant for the calibration of the tree.

2.2. Laboratory techniques

DNA was extracted from herbarium material or silica driedsamples using the DNeasy plant extraction kit (Qiagen, Hilden,Germany) according to the manufacturer’s protocol. Amplificationswere performed using 1.5 mM buffer, 0.625 mM MgCl2, 0.2 mMdNTPs, 0.05 U/ll Taq DNA polymerase (Amersham Biosciences),0.325 lM primer (ITS-A and ITS-B [Blattner, 1999] for nuclear ITSand atpB-F2 and atpB-R5 [Manen et al., 1994] for the plastidatpB-rbcL intergenic spacer) and 5 ng/ll DNA template. PCR pro-files consisted of 33 cycles of 94 �C for 1 min, 50–55 �C for 1 min,and 72 �C for 2–3 min. PCR products were cleaned using the PCRpurification kit (Qiagen) and sequenced in both directions withthe PCR primers using BigDye 3.1 Terminator chemistry (AppliedBioystems, Foster City, California). The resulting products wereseparated on an ABI PRISM 3100 automated sequencing systems(PE Biosystems).

2.3. Data analysis

Sequences were initially aligned using Clustal X (vers. 1.81;Thompson et al., 1997) and then adjusted manually. The sequencedata from the two genomes were analysed separately to test forcongruence. The congruence assumption was rejected if trees fromthe two data sets contained incongruent groupings supported by>70% bootstrap (Mason-Gamer and Kellogg, 1996). The 70% is arbi-trary, but has been widely used (e.g., Moline et al., 2007). As noincongruent nodes were retrieved, the nuclear and chloroplast datasets were combined in a total evidence approach (Johnson and Sol-tis, 1998; Wiens, 1998). The alignment of the combined data set isavailable as supplement.

Maximum likelihood analyses for the combined data set wereperformed using PAUP4.0 (Swofford, 2002). A GTR+C+I modelwas found to be the best-fit substitution model determined byusing AIC as implemented in MODELTEST 3.6 (Posada and Crandall,1998). Using the model parameters suggested by MODELTEST, a

heuristic search with 100 random-addition-sequence replicates,TBR branch swapping and steepest descent option in effect wasconducted. The same options were used for ML bootstrap analyseswith 100 bootstrap replicates. We tested the hypothesis that Mac-aronesian and Eritreo–Arabian taxa are not sister to each other, bycomparing the maximum likelihood topology with a tree in whichthese lineages were constrained not to be sister to each other. Aone-tailed SH test (Shimodaira and Hasegawa, 1999) with a testdistribution generated by using 1000 bootstrap replicates with fulloptimisation was conducted as implemented in PAUP4.0. The para-metric bootstrap procedure (Goldman et al., 2000; Stefanovic andOlmstead, 2004) included the determination of ML parametersfor the described constrained topologies. Based on these parame-ters, 99 simulated data sets were created using Seq-Gen (Rambautand Grassly, 1997). The simulated data sets were analysed usingmaximum parsimony with closest sequence addition, Multreeson and TBR branch swapping, testing for significant differences inlengths between the constrained tree as null hypothesis and theoptimal tree.

2.4. Molecular dating

To date the divergence events in Campylanthus a relaxed molec-ular clock was used. The dating analyses were conducted using thenrITS data set, because this allowed the inclusion of sequences ofAragoa, the sister group of Plantago, for which the chloroplast mar-ker was not available. To test for strict clock-like evolution of thenrITS sequences, a likelihood ratio test was performed by compar-ing the scores of ML trees with and without a molecular clock en-forced (Felsenstein, 1981; Sanderson, 1998; Nei and Kumar, 2000).

The Bayesian dating method (Thorne et al., 1998; Thorne andKishino, 2002) uses a probabilistic model to describe the changein evolutionary rate over time and uses the Markov chain MonteCarlo (MCMC) procedure to derive the posterior distribution ofrates and time. It allows multiple calibration points and providesdirect credibility intervals for estimated divergence times and sub-stitution rates. To compare results yielded by different methods,we used two programs, BEAST 1.4.8 (Drummond and Rambaut,2007) and MULTIDIVTIME (Thorne et al., 1998; Kishino et al.,2001; Thorne and Kishino, 2002). For BEAST analysis model param-eters (GTR+C+I, A/C: 0.9395, A/G: 1.68171, A/T: 1.8252, C/G:0.2043, C/T: 3.5625, C shape parameter 1.7796 and proportion ofinvariable sites 0.3089) for the nrITS data set as selected by AICwere used as initial values for Jeffreys priors. A relaxed clock modelwith an uncorrelated lognormal rate change was chosen. After tun-ing the operators using BEAST’s auto-optimisation option, analysesused random starting trees under the coalescent process and a spe-

Fig. 1. Map of northern Africa and Arabia showing the total distribution of Campylanthus (modified after Hjertson, 2003).

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ciation model following a birth–death process as tree prior, withtwo runs of 5 � 107 generations each, sampling every 103 genera-tions. Resulting posterior distributions for parameter estimateswere checked in Tracer 1.4.1 (Drummond and Rambaut, 2007)and maximum credibility trees, representing the maximum a pos-teriori topology, were calculated after removing burn-in with TreeAnnotator (version 1.4.7). The xml file is available as supplement.

We used MULTIDIVTIME following Thiv et al. (2006) and a step-by-step manual by Rutschmann (2004). This method requires afully resolved topology. Therefore, polytomies due to almostzero-branch lengths were resolved in accordance with a previousphylogenetic analysis based on morphological data (Figs. 6 and 4in Hjertson (1997, 2003), respectively). Model parameters for theF84+C model (Kishino and Hasegawa, 1989) were estimated usingthe module BASEML in PAML (Yang, 1997). Based on those maxi-mum likelihood branch lengths of the rooted tree together with avariance–covariance matrix of the branch length estimates weredetermined with ESTBRANCHES (Thorne et al., 1998). We usedMULTIDIVTIME to approximate the posterior distributions of sub-stitution rates and divergence times by using a multivariate nor-mal distribution of estimated branch lengths (provided here byESTBRANCHES) and running a MCMC procedure with the followingsettings for the prior distributions: 1.50 for both rttm and rttmsd,0.07 for both rtrate and rtratesd, 0.4 for both brownmean andbrownsd, and 42 million years ago (mya) for bigtime, which repre-sents the age of the stem node of the clade including Callitriche,Plantago, and Digitalis (Wikström et al., 2001). We ran the Markovchain for at least 103 cycles and collected one sample every 100 cy-cles, after an unsampled burn-in of 104 cycles. We repeated theanalyses in BEAST and MULTIDIVTIME twice using different initialconditions to assure convergence of the Markov chain and com-bined the results.

Geological calibration dates and fossil data were used formolecular dating estimates (e.g., Richardson et al., 2001; Forestet al., 2005). For BEAST and MULTIDIVTIME analyses two calibra-tion points were used. Plantago stauntonii is a species endemic tothe Pacific island of New Amsterdam (Tongatapu Islands), of whichthe geological age is known to be 0.5–0.7 mya (Rønsted et al.,2002). If we assume that speciation of the island taxon from itsmainland ancestor occurred following dispersal to the island, thenthe geological age of islands represents the maximum age of ende-mic taxa. There is no evidence that New Amsterdam is part of a hotspot system (Rønsted et al., 2002), in which an endemic speciescould have established earlier on an now-drowned island, beforedispersing to the current island, thus distorting the real age ofthe species (Heads, 2005). Therefore, we used the older age of0.7 mya as the upper bound for the crown group age of the cladeof P. stauntonii, P. debilis and P. spathulata. A minimum age forthe stem node age of Plantago was derived from pollen fossilsattributed to Plantago. Records extend to the Middle/Upper Mio-cene (Nagy, 1963; Gray, 1964; Muller, 1981). Dates of Plantago fos-sils were published by Naud and Suc (1975), (6.4 mya, France) andMohr (1984), (5.3–7.2 mya, Germany). These dates are also sup-ported by molecular dating using P. stauntonii, yielding an estimateof ca. 5.5 mya (Rønsted et al., 2002). Still, to our knowledge the old-est fossil record attributed to Plantago dates to the Sarmatian(Upper Middle-Miocene; 12.8–11.6 mya, Harzhauser and Piller,2004). This polyporate pollen was described as Plantaginacearum-pollis and resembles the one of the extant Plantago lanceolata L.(Nagy, 1963). Although Aragoa and Plantago share similar exinestructures (Bello et al., 2002) their pollen can be distinguished bynumber and shape of the apertures. Plantago is characterised by2–14-porate pollen (Saad, 1982) while Aragoa has tricolpate pollen(Nilsson and Hong, 1993). Despite the vast fossil record of Plantagowe cannot rule out the existence of older fossils. Accordingly, weused 11.6 mya as the lower bound for the stem node of Plantago.

2.5. Biogeographical analyses

A crucial initial step in cladistic biogeography is the definitionof the organisms’ area of distribution (Linder, 2001). We recogni-sed Macaronesia (= MA) including the Canary and Cape Verde Is-lands, and the three provinces of the Eritreo–Arabian subregionaccording to Takhtajan (1986): Somalo-Ethiopia, SE; South Ara-bia, SA and Socotra, SO. The distribution ranges of the specieswere based on Hjertson (1997, 2003) and are indicated in Fig3. Except for C. junceus and C. spinosus, all species of Campylan-thus are restricted to a single region. Campylanthus anisotrichus(SE), C. parviflorus (SE), the Omanian C. hajarensis and C. ramosiss-imus from Pakistan (for which no molecular sequence data wereavailable), likely do not effect the reconstruction of ancestralareas (see Section 4). Recent biogeographical analytical programstake into account the connectivity between areas. We used La-grange 2.0.1 (Ree and Smith, 2008) for the reconstruction of bio-geographic areas based on an ultrametric subtree of the BEASTconsensus topology including all Campylanthus accessions andGlobulariaceae. The two Globularia species were treated as a sin-gle terminal taxon because the exact patterns between theunsampled Poskea and the remaining Globularia species werenot considered. The distribution of Globulariaceae (SA, SE, SOand MA) reflects occurrences of Poskea in SA, SE and SO andGlobularia in SE and MA, however, not taking into account thedistribution of Globularia far outside that of Campylanthus. Still,similar results for Campylanthus were yielded when all taxa fromthe BEAST analysis were coded (not shown). The basal split wasdated to 13 mya as suggested by BEAST analysis. All combina-tions of areas were allowed in the adjacency matrix and baselinerates of dispersal and local extinction were estimated. Two mod-els were considered. The first model did not constrain links be-tween any areas throughout time. The second model includedtwo constraints. The aridification of the Sahara (about 7 mya,Schuster et al., 2006) was taken into account by reducing thesymmetrical dispersal rate between MA and the remaining areasto 0.1 in a time frame of 0–7.0 mya. Furthermore, the split be-tween Socotran and east African accessions of C. spinosus was re-garded as result of recent long-distance dispersal because theseareas have not been linked with each other in the Pliocene. In-stead, the Socotran archipelago was formerly connected to Arabia(15–18 mya; Fleitmann et al., 2004, Van Damme, 2009). There-fore, we regarded triplets with SO as unlikely ancestral areas,especially for basal nodes, and excluded SO–SA–SE, SO–SA–MAand SO–SE–MA, not SO–SE–SA–MA because this was coded forGlobularia, and limited the total number of areas to three. Dueto low temporal resolution the repeated closure of the Red Seacould not been modelled (see Section 4).

3. Results

The aligned sequence lengths were 646 bp (ITS1, 5.8 SrDNA,ITS2) and 523 bp (atpB-rbcL spacer), resulting in a total of1169 bp for the combined data set (including 1.9% of cells withmissing data), of which 332 were variable and 180 were potentiallyparsimony-informative.

The selected optimal model of sequence evolution for this com-bined data set was the general time reversible (GTR+C+I) model(Rodriguez et al., 1990): unequal base frequencies (A = 0.2656,C = 0.2252, G = 0.2431, T = 0.2661), six substitution types (A/C:1.3514, A/G: 1.7381, A/T: 1.1212, C/G: 0.3735, C/T: 4.2855), gammadistribution of rates among sites with alpha shape parameter0.8682 and proportion of invariable sites 0.4097. The analysis usingthese parameters yielded a ML tree with a log-likelihood score of�lnL = 4232.01 (Fig. 2).

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The combined data set strongly supports the monophyly ofCampylanthus with a bootstrap (= BS) of 100%. Within the genus,C. glaber from the Cape Verde Islands and the Canarian C. salsoloidesconstitute the sister group (BS 97%) to the eastern African–Arabianspecies (BS 85%). Within the latter clade, C. mirandae, C. pungens,and C. junceus/C. yemenensis (= C. pungens group; BS 89%) form aclade sister to a group including C. chascanifolius, C. sedoides, C.antonii/C. hubaishanii (= C. sedoides group, BS 98%) and C. somalien-sis. The two clades in turn are sister to a group including C. incanus/C. reconditus and C. spinosus (BS 82%).

The alternative hypothesis of the Macaronesian and all Eritreo–Arabian taxa not being sister groups resulted in a difference of thelog-likelihood of 4.47 in the SH test and in a difference of treelength of 2 steps in the parametric bootstrap analysis, neither testthus rejecting the alternative hypothesis (P = 0.173 and P = 0.41,respectively).

3.1. Molecular dating and biogeographical analyses

Enforcing the molecular clock resulted in a log-likelihood scoreof �lnL = 3754.77 for the nrITS data set (Table 1, Fig. 3). The com-

parison between clock and non-clock trees (�lnL = 3717.86) byapplying the likelihood ratio (LR) test significantly rejected clock-like evolution for the dataset (LR = 73.82; df = 27, P < 0.001). Theresults of the two runs using BEAST were very similar, and weretherefore combined. The same applied to the two runs using MUL-TIDIVTIME. The estimated mean ages and 95% highest posteriordensity intervals (HPD) are shown in Table 2. Although the ages in-ferred by BEAST are generally younger than those by MULTIDIV-TIME, both methods date the basal split within Campylanthus tothe Upper Miocene/Pliocene, with estimates between 2.00–8.08and 3.02–9.84 mya (node a, Table 2). A chronogram of one of thetwo runs made by BEAST is shown in Fig. 3.

The results of the biogeographical analyses are given in Table 2.Most relevant to the questions addressed in this paper are the re-sults for the divergence between the Macaronesian and the Erit-reo–African clades (node a in Fig. 3). Depending on theunderlying model, the geographical division with the highest prob-ability was either MA and SA/SE/SO (model 1) or MA and SA/SE(model 2). Among Arabian and east African species, the resultsare quite similar, however, larger incongruence between the mod-els was found in following cases. The highest probability for node c

Digitalis lutea Digitalis lutea

Veronica filiformisVeronica filiformis

Plantago majorPlantago major

Globularia punctataGlobularia punctata/repensrepens

C. glaber C. glaber 31403140

C. salsoloides C. salsoloides 34853485

C. incanus C. incanus 1072010720

C. reconditus C. reconditus 1065210652

C. spinosusC. spinosus 23742374

C. spinosus C. spinosus 2347723477

C. somaliensis C. somaliensis 55605560

C. chascaniflorus C. chascaniflorus 77317731

C. sedoides C. sedoides 94519451

C. antonii C. antonii 95349534

C. hubaishanii C. hubaishanii 11699116

C. mirandae C. mirandae 561561

C. pungens C. pungens 51385138

C. junceus C. junceus 60366036

C. yemenensis C. yemenensis 30763076

C. yemenensis C. yemenensis 75/22575/2250.01 substitutions/site0.01 substitutions/site

7070

98

9797

100

8585

8282

8484

100100

9898

8181

99999090

8989

8282

100100

Fig. 2. ML tree of Campylanthus based on combined nrITS and atpB-rbcL intergene sequences. ML-Bootstrap values (>50%) are on branches. Numbers following Campylanthus(= C.) species names refer to collection numbers shown in Table 1.

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in Fig. 3 was SE|SA, SE using model 2, whereas the node was opti-mised SE, SO|SA under model 1. The models also differed for node ffavouring a split between Arabia (SA|SA) or SA|SA, SE.

4. Discussion

4.1. Phylogeny

Our phylogenetic analysis using combined chloroplast and nu-clear data corroborate the monophyly of Campylanthus and the ba-sal bipartition between the Macaronesian clade of C. glaber and C.salsoloides and the lineage of the remaining species from easternAfrica, Arabia, and likely southern Asia. This is in accordance withthe morphological analysis by Hjertson (1997, 2003). We testedalternative scenarios using SH test and parametric bootstrap. TheSH test is a nonparametric test which is a recommended way ofassessing support when the number of candidate trees is small(Shimodaira, 2002) and parametric bootstrap is shown to be a sta-tistically sound method of evaluating different alternative topolog-ical hypotheses (Huelsenbeck et al., 1996; Goldman et al., 2000).Curiously, our parametric bootstrap and SH tests indicated thatalternative topologies cannot be rejected. Such trees placed thetwo Macaronesian species nested inside the Eritreo–Arabian taxaas sister to C. incanus, C. reconditus and C. spinosus. This could bedue to low phylogenetic signal, however, as the basal split betweenMacaronesian and Eritreo–Arabian Campylanthus gains high boot-strap and morphological (Hjertson, 1997, 2003) support, we favourthis topology as our working hypothesis.

Most of the Eritreo–Arabian clades show uniform geographicpatterns. Well-supported sister group to all Arabian species plusC. somaliensis is the eastern African and partly Socotran groupof C. incanus/C. reconditus/C. spinosus. Based on the commonoccurrence of adpressed hairs on the vegetative parts, this cladeshould also include the unsampled Somalian C. anisotrichus andC. parviflorus (Hjertson, 1997, 2003). Other morphological charac-ters support the incorporation of C. ramosissimus from Pakistanand C. hajarensis from Oman into the mainly Arabian C. pungensclade (Hjertson, 1997, 2003; Hjertson et al., 2008). The mono-phyly of the C. sedoides group including C. somaliensis is corrobo-rated by particular style shapes (Hjertson, 1997, 2003). Overall,the relationships among Eritreo–Arabian species of Campylanthusinferred from molecular data differ from those based on morpho-logical characters (Hjertson, 1997, 2003), and none of these cladesexactly match those in our study. Nonetheless, several taxonomicaffinities as discussed by Hjertson (1997, 2003) are corroboratedby our data, e.g., the close relationships between C. incanus andC. spinosus, and between C. pungens, C. junceus, C. yemenensis,and C. mirandae.

4.2. Vicariance hypothesis

The vicariance hypothesis (e.g., Axelrod, 1975; Bramwell, 1985)predicts that (i) Macaronesian and Eritreo–Arabian taxa should besister groups and (ii) that the age of their split should fall in theUpper Miocene to Pliocene, the period in which the aridificationof the Sahara took place, which, according to palaeoecological evi-dence, started at least 7 mya (Schuster et al., 2006). Both criteria

Plantago majorPlantago major

Globular ia repensGlobular ia repens

C. glaber C. glaber MAMAC. salsoloides C. salsoloides MAMA

C. incanus C. incanus SEC. recondi tus C. recondi tus SEC. spinosusC. spinosus SO SOC. spinosus C. spinosus SE

C. somal iensis C. somal iensis SESEC. chascani f lorus C. chascani f lorus SASAC. sedoides C. sedoides SA

C. antoni i C. antoni i SAC. hubaishani i C. hubaishani i SASA

C. mirandae C. mirandae SAC. pungens C. pungens SAC. junceus C. junceus SA, SESA, SEC. yemenensis C. yemenensis SASAC. yemenensis C. yemenensis SASA

Globular ia sal ic inaGlobular ia sal ic ina

Plantago uni f loraPlantago uni f lora

Plantago raoul i iPlantago raoul i iPlantago stauntoni iPlantago stauntoni iPlantago debi l isPlantago debi l isPlantago spathulataPlantago spathulata

Plantago atrataPlantago atrata

Plantago coronopusPlantago coronopusAragoa cupressinaAragoa cupressinaAragoa abiet inaAragoa abiet inaDigi ta l is luteaDigi ta l is lutea

max. 0.7 mya

min. 11.6 mya

0515 1020 mya

a

b

d

c

h

e

g

f

i

Fig. 3. Chronogram of Campylanthus of the Bayesian dating analysis using BEAST based on nrITS sequences. Lower case letters refer to nodes as in Table 2. Letters behindCampylanthus (= C.) species indicate defined areas of distribution (MA, Macaronesia; SE; Somalo-Ethiopia; SA, South Arabia; SO, Socotra). Arrows define calibration nodes withgiven ages. Gray bars indicate 95% HPD of age estimates.

612 M. Thiv et al. / Molecular Phylogenetics and Evolution 54 (2010) 607–616

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are met in Campylanthus. First, the split between the Macaronesianclade and the Eritreo–Arabian one is corroborated, and, second, thedivergence time of these groups is estimated to be 4.68 (2.00–8.07)and 6.15 (3.02–9.84) mya using BEAST and MULTIDIVTIME, respec-tively. Whereas the results of MULTIDIVTIME coincide with theproposed inception of climate change 7 mya, age estimates byBEAST are somewhat younger. All present day Campylanthus spe-cies inhabit dry regions and show several adaptations to theseenvironments (Hjertson, 1997, 2003). Possibly, ancestral, northernAfrican populations were already drought adapted and were ableto colonise drier niches within the sclerophyllous, evergreenwoodland belt before they became subsequently victim of progres-sive, extreme aridification. This could explain a possible delayedextinction, especially of sclerophyllous taxa in the Saharan belt.Additionally, also in agreement with the vicariance hypothesis,our biogeographic analyses indicated a basal split between wes-tern and eastern groups of Campylanthus encompassing Macarone-sia and east Africa/Arabia and possibly Socotra (Table 2). Thesefindings make the postulation of trans-Saharan long-distance dis-persal redundant.

The climatic shift across northern Africa since the Upper Mio-cene should have led to similarly vicariant trans-Saharan distribu-tion patterns in other plant groups. Such taxa were recentlyreviewed by Andrus et al. (2004). The evidence from phylogeneticanalyses of such taxa is ambiguous. A vicariance scenario is re-futed, for instance, for Tolpis Adanson (Asteraceae, Park et al.,2001; Moore et al., 2002) and Ceropegia L. (Apocynaceae, Bruyns,1985). Biogeographic relationships between Macaronesia and eastAfrica/Arabia do exist for Aeonium. Here, the east African A. leuco-blepharum Webb ex A. Rich. is deeply nested within the Macarone-sian clade (Mort et al., 2002) and has probably dispersed from westto east no later than in the Pliocene or Pleistocene (Kim et al., 2008;Thiv et al., in press), possibly facilitated by the presence of numer-ous small seeds. For other taxa, available data are consistent withthe vicariance hypothesis, for instance for Nanorrhinum Betsche(Plantaginaceae, Ghebrehiwet, 2000), Kleinia Mill. (Asteraceae, Pel-ser et al., 2007; M. Thiv, unpubl. data) and Dracaena (Marrero et al.,1998). The same applies to Camptoloma (Kornhall et al., 2001), inwhich sister taxa occur across the Sahara, and have links to south-ern Africa, as also shown for Plocama Ait. (Backlund et al., 2007)illustrating a pattern of the Rand flora (Sanchez-Meseguer et al.,2009). Even the present day distribution of Globularia in Macarone-sia/Mediterranean area/northern Europe and Poskea in Eritreo–

Arabia may reflect such a vicariance origin (Wagenitz, 2004; Al-bach et al., 2005).

4.3. Biogeographic history

The aridification of the Sahara may have divided a formerlywidespread ancestor of Campylanthus into a western and easterngroup. The western lineage is today represented by C. salsoloideson the Canary Islands and C. glaber on the Cape Verde Islands.The divergence time between these species in the Pleistocene–Pli-ocene (node b, Table 2) is much younger than the geological ages ofthe oldest Canarian island (Fuerteventura, 20.6 mya, Carracedoet al., 2002) and of Cape Verde island (Miocene, Mitchell-Thomé,1972; Rothe, 1982; Boekschoten and Manuputty, 1993). A vicari-ance scenario giving rise to a differentiation between the two Mac-aronesian species can be ruled out because there is no evidencethat these two volcanic archipelagos were ever connected witheach other (Carracedo et al., 2002). Accordingly, Campylanthusprobably colonised these islands via long-distance dispersal. Withthe data available it is not possible to determine whether the Mac-aronesian clade originated in the Canary Islands and dispersed tothe Cape Verde Islands, like Sonchus and Aeonium (Kim et al.,2008), or vice versa. Whatever the route among the island, theancestor must have come from the mainland. Close biogeographicrelationships between north-western Africa/Mediterranean on theone hand and the Canary islands on the other hand have been sug-gested for numerous plants. Phylogenetic analyses, e.g., of the Son-chus alliance (Asteraceae), dragon trees, Ixanthus Griseb.(Gentianaceae) or Macaronesian Crassulaceae–Sempervivoideae(Kim et al., 1996, 2008; Marrero et al., 1998; Thiv et al., 1999, inpress; Mort et al., 2002) corroborated the hypothesis that themainland served as source area for the island colonisation (Médailand Quézel, 1999; Sanmartín et al., 2008). A third explanation isthat C. salsoloides and C. glaber could have reached the islands inde-pendently from (north-)western Africa as shown for bryophytesand pteridophytes (Vanderpoorten et al., 2007), where this lineagelater became extinct.

The eastern clade of African–Arabian species of Campylanthus isgeographically structured, and these biogeographic patterns couldreflect another case of vicariance. The mostly African group of C.incanus, C. reconditus, and C. spinosus is sister to the remaining pri-marily Arabian taxa. Most of these species grow along the Gulf ofAden, the Somalia block and parts of the eastern Red Sea (Fig. 1).

Table 2Results of the Bayesian dating using BEAST and MULTIDIVTIME showing combined mean ages, 95% HPD (all in mya) of two runs, and the biogeographic reconstructions usingLagrange based on models 1 and 2 (see Section 2). Abbreviations follow Table 1, nodes refer to Fig. 3.

BEAST MULTIDIVTIME Lagrange model 1 Lagrange model 2

Node Mean 95% HPD Mean 95% HPD Split lnL Rel. prob. Split lnL Rel. prob.a 4.68 2.00–8.07 6.15 3.02–9.84 [SA, SE, SO|MA] �18.17 0.6402 [SA, SE|MA] �18.82 0.6279

[SA, SE|MA] �19.13 0.2452 [SE|MA] �19.68 0.265b 1.16 0.16–2.61 3.12 0.59–6.76 [MA|MA] �17.72 1 [MA|MA] �18.36 1c 3.34 1.40–5.65 4.90 2.10–8.47 [SE, SO|SA] �18.41 0.5026 [SE|SA, SE] �19.14 0.4581

[SE|SA, SE] �19.45 0.1788 [SE|SA] �19.62 0.2814[SO|SA, SE] �19.52 0.1665 [SE|SE] �19.97 0.2002[SE|SA] �20.36 0.0718

d 1.45 0.35–2.90 3.33 0.95–6.70 [SE, SO|SE] �17.97 0.7797 [SE|SE] �19.04 0.5036[SE|SE] �19.39 0.189 [SE, SO|SE] �19.14 0.4583

e 0.99 0.00–0.32 0.83 0.02–3.12 [SE|SO] �17.72 1 [SE|SO] �18.36 1f 2.34 1.00–4.02 3.73 1.40–7.06 [SA|SA] �18.29 0.5693 [SA|SA, SE] �19.26 0.4073

[SA|SA, SE] �18.86 0.3221 [SA|SA] �19.59 0.2911[SE|SE] �20.41 0.1286[SA|SE] �21.17 0.0602

g 1.38 0.49–2.45 2.64 0.82–5.58 [SA|SA] �17.87 0.8605 [SA|SA] �18.74 0.6848[SA|SA, SE] �19.72 0.1365 [SA|SA, SE] �19.56 0.3004

h 1.03 0.17–2.06 1.48 0.14–3.99 [SA|SA] �17.98 0.7749 [SA|SA] �18.86 0.6042[SA|SA, SE] �19.25 0.2175 [SA|SA, SE] �19.35 0.3687

i 0.81 0.23–1.57 1.80 0.38–4.40 [SA|SE] �17.72 1 [SA|SE] �18.36 1

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Their basal split probably falls into the Lower Pliocene/Upper Mio-cene (node c: Table 2). During this period repeated land connec-tions between Africa and Arabia enabled several African mammalspecies to colonise Central Europe. This migration route was closedwhen oceanic rifting opened the south-central Red Sea and thepropagation of the Sheba Ridge widened the Gulf of Aden (Steinin-ger et al., 1985; Bosworth et al., 2005). These events correspond intime to the Messinian salinity crisis of the Mediterranean basin6 mya and might have separated the primarily African and Arabiangroups of Campylanthus. This pattern is indicated by splits betweenSA and SE in Lagrange analyses (Table 2: node c), but show ratherlow likelihoods under both models. Although Lagrange indicates adivision between SE and SA for S. somaliensis and its sister (node i),vicariance is unlikely because the estimated age is much youngerthan the geological separation.

Alternatively, all Eritreo/Arabian disjunctions could also be ex-plained with dispersal events. In the dispersal scenario either east-ern Africa or Arabia was the area of origin. If eastern Africa was thearea of origin (Table 2: node c, model 2), then the southern ArabianC. sedoides and C. pungens groups, irrespective of C. junceus, are theresult of either two independent (Table 2: nodes g and i) or a singledispersal event from eastern Africa (Table 2: node f, model 1). Inthis scenario, the east African distribution of C. somaliensis is morelikely explained by secondary back-dispersal from Arabia becauseno evidence for vicariance is coeval with the age determination.If the entire group is postulated to have originated in Arabia, thenAfrica was colonised twice (Fig. 3: nodes d and i).

Under a parsimony criterion, however, these dispersalist sce-narios for the African–Arabian taxa are less likely since they re-quire more steps than a vicariance explanation. Even if thevicariance hypothesis is accepted, long-distance dispersals musthave been involved where no geographic links can be assumed.This is the case for C. junceus which occurs in Arabia and Africa,for the Sindian C. ramossisimus from Arabia to Pakistan and for C.spinosus from east Africa to Socotra. The estimated age of the Soco-tran accession of up to 3.12 mya clearly falls within the geologicalage of the island group of at least 15 mya (Fleitmann et al., 2004)and is younger than that of other Socotran plants (Aerva Forsk.,Thiv et al., 2006), which seems plausible regarding the conspecifityof African and Socotran C. spinosus.

In conclusion, our data support the hypothesis for a vicariantorigin of the disjunct distribution of Campylanthus between Maca-ronesia and east Africa–Arabia. This may be the result of the aridi-fication of the Sahara in the Upper Miocene and Pliocene.Vicariance resulting from climatic changes, as suggested forCampylanthus, may be a much more common process then hithertoassumed.

Acknowledgments

The authors thank Gerald M. Schneeweiss (Vienna, Austria),Merijn Bos (Stuttgart, Germany) and Arno Wörz (Stuttgart, Ger-many) for valuable comments on the paper, Frank Rutschmann(Bern, Switzerland), Daniele Silvestro (Frankfurt, Germany) andAndreas Franzke (Heidelberg, Germany) for helpful advice forusing MULTIDIVTIME, Lagrange, and BEAST, respectively, VeronikaWähnert (Freiburg, Germany), Johanna Eder (Stuttgart, Germany)and Barbara Mohr (Berlin, Germany) for the evaluation of the fossilrecord, Norbert Kilian (Berlin, Germany) for plant material and lit-erature, Mohamed Ali Hubaishan (AREA Research Station Mukalla),Ahmed Said Sulaiman (EPA Socotra), Said Masood Awad Al-Gareiri(Dept. Agriculture Socotra), and Mohamed El-Mashjary (EPA Sa-naa; all Yemen) for support of the field work on Socotra. The fieldwork was conducted as part of the BIOTA Yemen Project funded bythe German Ministry for Research and Education (BMBF). Thisstudy was supported by Grants of the German Research Founda-

tion (DFG, Th830/1-1) and the Claraz-Schenkung (Switzerland) tothe first author.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2009.10.009.

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